Modified Guide RNAs

ABSTRACT

This disclosure relates to modified single and dual guide RNAs having improved in vitro and in vivo activity in gene editing methods.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Dec. 2, 2022, is named 01155-0004-01US_SequenceListing_ST26 and is 509,585 bytes in size.

This application is a Divisional of U.S. application Ser. No. 16/434,512, which was filed on Jun. 7, 2019, which is a Continuation of International Application No. PCT/US2017/065306, which was filed on Dec. 8, 2017, and which claims the benefit of priority to U.S. Provisional Application No. 62/431,756, which was filed on Dec. 8, 2016, all of which are incorporated by reference in their entirety.

This disclosure relates to the field of gene editing using CRISPR/Cas systems, a part of the prokaryotic immune system that recognizes and cuts exogenous genetic elements. The CRISPR/Cas system relies on a single nuclease, termed CRISPR-associated protein 9 (Cas9), which induces site-specific breaks in DNA. Cas9 is guided to specific DNA sequences by small RNA molecules termed guide RNA (gRNA). Guide RNA comprises trRNA (also known as tracrRNA) and crisprRNA (crRNA). The trRNA and crRNA may be contained within a single guide RNA (sgRNA) or in two separate RNA molecules of a dual guide RNA (dgRNA). Cas9 in combination with trRNA and crRNA or an sgRNA is termed the Cas9 ribonucleoprotein complex (RNP).

Oligonucleotides, and in particular RNA, are sometimes degraded in cells and in serum by endonuclease or exonuclease cleavage. Improved methods and compositions for preventing such degradation, improving stability of gRNAs and enhancing gene editing efficiency is desired, especially for therapeutic applications.

SUMMARY

In some embodiments, therapeutic genome editing tools are provided comprising modified guide RNAs. The modified guide RNAs described herein may improve the stability of the guide RNA and the guide RNA/Cas9 complex and improve the activity of Cas9 (e.g., SpyCas9 and equivalents) to cleave target DNA. In some embodiments, the guide RNA is an sgRNA. In some embodiments, the guide RNA is a dgRNA. In some embodiments, the guide RNA is a tracrRNA. In some embodiments, the guide RNA is a crRNA.

The guide RNAs described herein comprise at least one modified nucleotide. Modifications may include 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxyethyl) (2′-O-moe), 2′-fluoro (2′-F), phosphorothioate (PS) bond between nucleotides, G-C substitutions, and inverted abasic linkages between nucleotides and equivalents thereof. Embodiments of the invention include:

In some embodiments, a single guide RNA (sgRNA) is encompassed comprising a 5′ end modification and one or more modification in one or more of: the upper stem region; the hairpin 1 region; and the hairpin 2 region, wherein the 5′ end modification comprises at least two phosphorothioate linkages within the first seven nucleotides at the 5′ end of the 5′ terminus. In some instances, the modification is a 2′-O-methyl (2′-O-Me) modified nucleotide. In some embodiments, the modification is a 2′-fluoro (2′-F) modified nucleotide.

In some embodiments, the sgRNA comprises modifications at US1 to US12 and/or a modification at H1-1 and/or a modification in H2-1. In some embodiments, the sgRNA comprises modifications at H1-1 to H1-12 and/or H2-1 to H2-15. In some embodiments, the sgRNA comprises one or more modifications in each of the upper stem region, the hairpin 1 region, and the hairpin 2 region. In some embodiments, the sgRNA comprises a modified nucleotide between hairpin 1 and hairpin 2 regions. In some embodiments, the sgRNA comprises a modification in the lower stem region.

In some embodiments, the sgRNA comprises a modification at the 5′ terminus and/or the 3′ terminus. In some embodiments, the sgRNA comprises a 3′ end modification in the 3′ terminus. In some embodiments, the sgRNA comprises modifications on at least two of the last four nucleotides at the 3′ end of the 3′ terminus. In some embodiments, the sgRNA comprises a 5′ end modification in the 5′ terminus. In some embodiments, the sgRNA comprises modifications on at least two of the first four nucleotides at the 5′ end of the 5′ terminus. In some embodiments, the sgRNA comprises a 3′ end modification in the 3′ terminus and a 5′ end modification in the 5′ terminus. In some embodiments, the sgRNA comprises modifications on at least two of the last four nucleotides at the 3′ end of the 3′ terminus and on at least two of the first four nucleotides at the 5′ end of the 5′ terminus. In some instances, these modifications are 2′-O-Me, 2′-F, 2′-O-moe, or phosphorothioate (PS) bonds linking the nucleotides. In some embodiments, the sgRNA comprises PS bonds between at least two of the last four nucleotides at the 3′ end of the 3′ terminus and/or at least two of the first four nucleotides at the 5′ end of the 5′ terminus. In some instances, the sgRNA comprises 5′ terminus and 3′ terminus with more than one modification as described herein, such as, with PS bonds and 2′-O-Me modifications.

In some embodiments, the sgRNA comprises a modification in the bulge region. In some embodiments, 50% of the nucleotides in the bulge region are modified, wherein the modification is 2′-O-Me or 2′-F.

In some embodiments, the sgRNA comprises a modification in the nexus region. In some embodiments, the sgRNA comprises modifications at N15, N16, N17, and/or N18 in the nexus region, wherein the modification is 2′-O-Me or 2′-F. In some instances, N16, N17, and N18 are linked with PS bonds.

In some embodiments, the sgRNA comprises at least the first three nucleotides at the 5′ end of the 5′ terminus, and the last three nucleotides at the 3′ end of the 3′ terminus are modified.

In some embodiments, the sgRNA comprises modifications at the 3′ terminus and/or 5′ terminus. In some instances, the first four nucleotides at the 5′ end of the 5′ terminus, and the last four nucleotides at the 3′ end of the 3′ terminus are linked with phosphorothioate (PS) bonds. In some embodiments, the 5′ and 3′ modification comprises 2′-O-Me or 2′-O-moe. In some embodiments, the 5′ and 3′ modification comprises 2′-F. In some embodiments, the 5′ and/or 3′ modification comprises PS bonds linking nucleotides. In some embodiments, the 5′ and/or 3′ modification comprises one or more of 2′-O-Me, 2′-O-moe, 2′-F, and PS bonds linking nucleotides.

In some embodiments, the sgRNA comprises modifications at the first four nucleotides at the 5′ end of the 5′ terminus and the last four nucleotides at the 3′ end of the 3′terminus. In some instances, these modifications are linking PS bond (i.e., PS bonds that link the first four and last four nucleotides). In some embodiments, the sgRNA further comprises 2′-O-Me modifications at the first three nucleotides at the 5′ end of the 5′ terminus and the last three nucleotides at the 3′ end of the 3′ terminus.

In some embodiments, the sgRNA comprises modifications at the first four nucleotides at the 5′ end of the 5′ terminus and the last four nucleotides at the 3′ end of the 3′ terminus, wherein the modifications are at least PS bonds linking the four nucleotides, and further wherein the first three nucleotides at the 5′ end of the 5′ terminus and the last three nucleotides at the 3′ end of the 3′ terminus comprise 2′-O-Me, 2′-O-moe, or 2′-F modifications.

In some embodiments, the sgRNA comprises modifications LS1, LS6, LS7, LS8, LS11, and LS12, wherein the modification is 2′-O-Me or 2′-F.

In some embodiments, the sgRNA comprises modifications at each of the nucleotides in the bulge region, wherein the modification is 2′-O-Me or 2′-F.

In some embodiments, the sgRNA comprises modifications at each of the nucleotides in the upper stem region, wherein the modification is 2′-O-Me or 2′-F.

In some embodiments, the sgRNA comprises modifications at each of the nucleotides in the hairpin 1 region, wherein the modification is 2′-O-Me or 2′-F.

In some embodiments, the sgRNA comprises modifications at each of the nucleotides in the hairpin 2 region, wherein the modification is 2′-O-Me or 2′-F.

In some embodiments, an sgRNA is encompassed comprising 2′-O-Me modified nucleotides at the following positions:

-   -   a. the first three nucleotides at the 5′ end of the 5′ terminus;     -   b. LS1, LS6, LS7, LS8, LS11, and/or LS12 in the lower stem         region;     -   c. B1 and/or B2 in the bulge region;     -   d. each nucleotide in the upper stem region;     -   e. N16, N17, and/or N18 in the nexus region;     -   f. each nucleotide in the hairpin 1 region;     -   g. each nucleotide in the hairpin 2 region; and     -   h. the last four nucleotides at the 3′ end of the 3′ terminus.         In some embodiments, B3-B6 are modified with 2′-O-Me. In some         instances, the sgRNA further comprising three phosphorothioate         (PS) bonds linking the first four nucleotides at the 5′ end of         the 5′ terminus and three PS bonds linking the last four         nucleotides at the 3′ end of the 3′ terminus. In some         embodiments, the sgRNA comprises 2′-F modifications at LS9 and         LS10. In some embodiments, the sgRNA comprises 2′F modifications         at N15, N16, N17, and N18. In some embodiments, the sgRNA         comprises 2′F modifications at H2-9, H2-10, H2-11, H2-12, H2-13,         H2-14, and H2-15. In some embodiments, the sgRNA comprises 2′F         modifications at the second to last, third to last, and fourth         to last nucleotides at the 3′ end of the 3′ terminus.

In some embodiments, an sgRNA is encompassed comprising 2′-F modified nucleotides at the following positions:

-   -   a. LS9 and LS10 in the lower stem region;     -   b. N15, N16, N17, and N18 in the nexus region; and     -   c. H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15 in the         hairpin 2 region.         In some embodiments, the sgRNA comprises 2′-F modified         nucleotides at the second to last, third to last, and fourth to         last nucleotides at the 3′ terminus. In some embodiments, the         sgRNA comprises three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ end of the 5′ terminus and         three PS bonds linking the last four nucleotides at the 3′ end         of the 3′ terminus. In some embodiments, the sgRNA comprises         2′-O-Me or 2′-F modified nucleotides at the first three         nucleotides at the 5′ end of the 5′ terminus, and 2′-O-Me or         2′-F modified nucleotides at three of the last four nucleotides         at the 3′ end of the 3′ terminus.

In some embodiments, an sgRNA is encompassed comprising

-   -   a. 2′-O-Me modified nucleotides at the first three nucleotides         at the 5′ end of the 5′ terminus;     -   b. Optional 2′-O-Me modified nucleotides at LS1 and/or LS6;     -   c. 2′-O-Me modified nucleotides at US1-US12;     -   d. 2′-O-Me modified nucleotides at H1-1-H1-12;     -   e. Optional 2′-O-Me modified nucleotide between Hairpin 1 and         Hairpin 2;     -   f. 2′-O-Me modified nucleotides at H2-1-H2-15; and     -   g. 2′-O-Me modified nucleotides at the last four nucleotides at         the 3′ end of the 3′ terminus; and optionally         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ end of the 5′ terminus and         three PS bonds linking the last four nucleotides at the 3′ end         of the 3′ terminus.

In some embodiments, an sgRNA is encompassed comprising:

-   -   a. 2′-O-Me modified nucleotides at the first three nucleotides         at the 5′ end of the 5′ terminus;     -   b. 2′-F modified nucleotides at LS1-LS6;     -   c. 2′-O-Me modified nucleotides at US1-US12;     -   d. 2′-O-Me modified nucleotides at H1-1-H1-12;     -   e. a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin         2;     -   f. 2′-O-Me modified nucleotides at H2-1-H2-15; and     -   g. 2′-O-Me modified nucleotides at the last four nucleotides at         the 3′ end of the 3′ terminus; and optionally         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ end of the 5′ terminus and         three PS bonds linking the last four nucleotides at the 3′ end         of the 3′ terminus.

In some embodiments, an sgRNA is encompassed comprising:

-   -   a. 2′-O-Me modified nucleotides at the first three nucleotides         at the 5′ terminus;     -   b. 2′-F modified nucleotides at LS2-LS5;     -   c. 2′-O-Me modified nucleotides at LS1 and LS6;     -   d. 2′-O-Me modified nucleotides at US1-US12;     -   e. 2′-O-Me modified nucleotides at H1-1-H1-12;     -   f. a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin         2;     -   g. 2′-O-Me modified nucleotides at H2-1-H2-15; and     -   h. 2′-O-Me modified nucleotides at the last four nucleotides at         the 3′ terminus and optionally         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ end of the 5′ terminus and         three PS bonds linking the last four nucleotides at the 3′ end         of the 3′ terminus.

In some embodiments, a sgRNA is encompassed comprising:

-   -   a. 2′-O-Me modified nucleotides at the first three nucleotides         at the 5′ terminus;     -   b. 2′-O-Me modified nucleotides at US1-US12;     -   c. 2′-O-Me modified nucleotides at LS7, LS8, LS11, and LS12;     -   d. 2′-O-Me modified nucleotides at H1-1-H1-12;     -   e. a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin         2;     -   f. 2′-O-Me modified nucleotides at H2-1-H2-15; and     -   g. 2′-O-Me modified nucleotides at the last four nucleotides at         the 3′ terminus, and optionally further comprising three         phosphorothioate (PS) bonds linking the first four nucleotides         at the 5′ end of the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ end of the 3′ terminus.

In some embodiments, a sgRNA is encompassed comprising:

-   -   a. 2′-O-Me modified nucleotides at the first three nucleotides         at the 5′ terminus;     -   b. 2′-O-Me modified nucleotides at US1-US12;     -   c. 2′-O-Me modified nucleotides at LS7, LS8, LS11, and LS12;     -   d. 2′-F modified nucleotides at LS9 and LS10;     -   e. 2′-O-Me modified nucleotides at H1-1-H1-12;     -   f. a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin         2;     -   g. 2′-O-Me modified nucleotides at H2-1-H2-15; and     -   h. 2′-O-Me modified nucleotides at the last four nucleotides at         the 3′ terminus, and optionally further comprising three         phosphorothioate (PS) bonds linking the first four nucleotides         at the 5′ end of the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ end of the 3′ terminus.

In some embodiments, an sgRNA is encompassed comprising:

-   -   a. 2′-O-Me modified nucleotides at the first three nucleotides         at the 5′ terminus;     -   b. 2′-O-Me modified nucleotides at US1-US12;     -   c. 2′-O-Me modified nucleotides at LS8, LS10, and LS12;     -   d. 2′-O-F modified nucleotides at LS7, LS9, and LS11;     -   e. 2′-O-Me modified nucleotides at H1-1-H1-12;     -   f. a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin         2;     -   g. 2′-O-Me modified nucleotides at H2-1-H2-15; and     -   h. 2′-O-Me modified nucleotides at the last four nucleotides at         the 3′ terminus, and optionally         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ end of the 5′ terminus and         three PS bonds linking the last four nucleotides at the 3′ end         of the 3′ terminus.

In some embodiments, a sgRNA is encompassed comprising:

-   -   a. 2′-O-Me modified nucleotides at the first three nucleotides         at the 5′ terminus;     -   b. 2′-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11, and         LS12     -   c. 2′-O-Me modified nucleotides at US1-US12;     -   d. 2′-O-Me modified nucleotides at H1-1-H1-12;     -   e. a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin         2;     -   f. 2′-O-Me modified nucleotides at H2-1-H2-15; and     -   g. 2′-O-Me modified nucleotides at the last four nucleotides at         the 3′ terminus, and optionally         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ end of the 5′ terminus and         three PS bonds linking the last four nucleotides at the 3′ end         of the 3′ terminus

In some embodiments, a sgRNA is encompassed comprising:

-   -   a. 2′-O-Me modified nucleotides at the first three nucleotides         at the 5′ terminus;     -   b. 2′-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11, and         LS12;     -   c. 2′-F modified nucleotides at LS9 and LS10;     -   d. 2′-O-Me modified nucleotides at US1-US12;     -   e. 2′-O-Me modified nucleotides at H1-1-H1-12;     -   f. a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin         2;     -   g. 2′-O-Me modified nucleotides at H2-1-H2-15; and     -   h. 2′-O-Me modified nucleotides at the last four nucleotides at         the 3′ terminus, and optionally         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ end of the 5′ terminus and         three PS bonds linking the last four nucleotides at the 3′ end         of the 3′ terminus.

In some embodiments, a sgRNA is encompassed comprising:

-   -   a. 2′-O-Me modified nucleotides at the first three nucleotides         at the 5′ end of the 5′ terminus;     -   b. 2′-O-Me modified nucleotides at US1-US12;     -   c. 2′-O-Me modified nucleotides at H1-1-H1-12;     -   d. a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin         2;     -   e. 2′-O-Me modified nucleotides at H2-1-H2-8;     -   f. 2′-F modified nucleotides at H2-9-H2-15;     -   g. 2′-F modified nucleotides at the second from last, third from         last, and fourth from last nucleotide at the 3′ terminus; and     -   h. a 2′-O-Me modified nucleotide at the last nucleotide at the         3′ terminus, and optionally         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ end of the 5′ terminus and         three PS bonds linking the last four nucleotides at the 3′ end         of the 3′ terminus.

In some embodiments, a sgRNA is encompassed comprising:

-   -   a. 2′-O-Me modified nucleotides at the first three nucleotides         at the 5′ end of the 5′ terminus;     -   b. 2′-O-Me modified nucleotides at US1-US12;     -   c. 2′-O-Me modified nucleotides at H1-2, H1-4, H1-6, H1-8,         H1-10, and H1-12;     -   d. 2′-F modified nucleotides at H1-1, H1-3, H1-5, H1-7, H1-9,         and H1-11;     -   e. a 2′-F modified nucleotide between Hairpin 1 and Hairpin 2;     -   f. 2′-F modified nucleotides at H2-2, H2-4, H2-6, H2-8, H2-10,         H2-12; and H2-14;     -   g. 2′-O-Me modified nucleotides at H2-1, H2-3, H2-5, H2-7, H2-9,         H2-11; H2-13, and H2-15;     -   h. 2′-F modified nucleotides at the second from last, and fourth         from last nucleotide at the 3′ terminus; and     -   i. 2′-O-Me modified nucleotide at the third from last and last         nucleotide at the 3′ end of the 3′ terminus, and optionally         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ end of the 5′ terminus and         three PS bonds linking the last four nucleotides at the 3′ end         of the 3′ terminus.

In some embodiments, a sgRNA is encompassed comprising:

-   -   a. 2′-O-Me modified nucleotides LS8, LS10, LS12, H1-2, H1-4,         H1-6, H1-8, H1-10, H1-12, H2-1, H2-3, H2-5, H2-7, H2-9, H2-11,         H2-13, and H2-15; and     -   b. 2′-F modified nucleotides at LS7, LS9, LS11; H1-1, H1-3,         H1-5, H1-7, H1-9, H1-11, H1-13, H2-2, H2-4, H2-6, H2-8, H2-10,         H2-12, and H2-14, and optionally         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ end of the 5′ terminus and         three PS bonds linking the last four nucleotides at the 3′ end         of the 3′ terminus; and optionally further comprising:     -   c. 2′-O-Me modified nucleotides at the last and third to last         nucleotide at the 3′ end of the 3′ terminus; and/or     -   d. 2′-F modified nucleotides at the second to last, fourth to         last, and/or last nucleotide at the 3′ end of the 3′ terminus.

In some embodiments, a sgRNA is encompassed comprising the nucleic acids of any of SEQ ID Nos: 228-353, including the modifications of Table 4. In some embodiments, a sgRNA is encompassed comprising any of SEQ ID Nos: 228-332, including the modifications of Table 4. In some embodiments, an sgRNA is encompassed comprising any of SEQ ID Nos: 235-240, 265-285, and 309-329, including the modifications of Table 4. In some embodiments, an sgRNA is encompassed comprising SEQ ID No: 240. In some embodiments, a sgRNA is encompassed comprising SEQ ID No. 240, including the modifications of Table 4. In some embodiments, a sgRNA is encompassed comprising SEQ ID No: 242. In some embodiments, a sgRNA is encompassed comprising SEQ ID No: 358. In additional embodiments, a sgRNA comprising nucleic acids having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to the nucleic acids of any one of SEQ ID Nos: 235-240, 265-285, and 309-329, wherein the modification at each nucleotide of the sgRNA that corresponds to a nucleotide of the reference sequence identifier in Table 4, is identical to or equivalent to the modification shown in the reference sequence identifier in Table 4, optionally further comprising three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ end of the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ end of the 3′ terminus. In some embodiments, the sgRNA further comprises at least three PS bonds linking the nucleotides in the hairpin 1 region. In some embodiments, the sgRNA further comprises at least three PS bonds linking the nucleotides in the hairpin 2 region. In some embodiments, the sgRNA further comprises at least three PS bonds linking the nucleotides in the upper stem region. In some embodiments, the sgRNA forms a ribonucleoprotein complex with S. pyogenes Cas9.

FIGURE LEGENDS

FIG. 1 shows percent editing as measured by next-generation sequence (NGS) of mouse transthyretin (TTR) gene following transfection of Neuro2A cells with modified crRNAs together with Cas9 mRNA and unmodified trRNA (TR000002).

FIG. 2 shows percent editing as measured by NGS of mouse TTR gene following transfection of Neuro2A cells with modified trRNAs together with unmodified crRNA (CR000686) and Cas9 mRNA.

FIG. 3 shows percent editing as measured by NGS of mouse TTR gene following transfection of Neuro2A cells with Cas9 mRNA and crRNAs and trRNAs having G-C pairings not found in parental sequences.

FIG. 4 shows percent editing as measured by NGS of mouse TTR gene following transfection of Neuro2A cells with modified crRNAs and trRNAs together with Cas9 mRNA. Standard deviations follow the value.

FIG. 5 shows percent editing as measured by NGS of mouse TTR gene following transfection of Neuro2A cells with modified sgRNAs together with Cas9 mRNA.

FIG. 6 shows percent editing as measured by NGS of mouse TTR gene following transfection of Neuro2A cells with modified crRNAs and unmodified trRNA (TR000002) together with Cas9 mRNA. The asterisk denotes a dual guide that for technical reasons did not show activity in this experiment. This dual guide was tested again in the experiment represented in FIG. 9 , in which it showed editing activity.

FIG. 7 shows percent editing as measured by NGS of mouse TTR gene following transfection of Neuro2A cells with unmodified crRNA (CR000686) and modified trRNAs together with Cas9 mRNA.

FIG. 8 shows percent editing as measured by NGS of mouse TTR gene following transfection of Neuro2A cells with Cas9 mRNA and crRNA and trRNA pairings with G-C pairings or G-U mismatches not found in the parental sequences.

FIG. 9 shows percent editing as measured by NGS of mouse TTR gene following transfection of Neuro2A cells with modified crRNAs and modified trRNAs together with Cas9 mRNA. Standard deviations follow the value.

FIG. 10 shows percent editing as measured by NGS of mouse TTR gene following transfection of Neuro2A cells with modified sgRNAs together with Cas9 mRNA.

FIG. 11 shows percent editing as measured by NGS of mouse Factor VII (FVII) gene following transfection of Neuro2A cells with modified sgRNAs together with Cas9 mRNA.

FIGS. 12A and 12B show percent editing as measured by NGS of mouse TTR (FIG. 12A) or FVII (FIG. 12B) following transfection of Neuro2A cells with modified crRNAs and unmodified trRNA together with Cas9 mRNA.

FIGS. 13A and 13B shows percent editing as measured by NGS of mouse TTR (FIG. 13A) or FVII (FIG. 13B) following transfection of Neuro2A cells with modified trRNAs and unmodified crRNA together with Cas9 mRNA.

FIGS. 14A, 14B, 14C, and 14D show interferon alpha (IFN-alpha, 14A), interleukin 6 (IL-6, 14B), monocyte chemotactic protein 1 (MCP-1, 14C), and tumor necrosis factor alpha (TNF-alpha, 14D) levels in serum after in vivo administration of LNPs comprising Cas9 mRNA and sgRNAs.

FIGS. 15A, 15B, and 15C show in vivo results following administration of LNPs comprising Cas9 mRNA and sgRNAs. FIG. 15A shows percentage of total editing in liver. FIG. 15B shows serum TTR levels. FIG. 15C shows the mean and standard deviation for the results of FIG. 15A. FIG. 15D summarizes modifications to the G000209 sgRNA (SEQ ID NO: 228). FIG. 15E summarizes modifications to the G000267 sgRNA (SEQ ID NO: 234). In FIGS. 15D and 15E, the nucleotides in bold are 2′-O-Me modified.

FIGS. 16A, 16B, 16C, and 16D show interferon alpha (IFN-alpha, 16A), tumor necrosis factor alpha (TNF-alpha, 16B), interleukin 6 (IL-6, 16C), and monocyte chemotactic protein 1 (MCP-1, 16D) levels in serum after in vivo administration of LNPs comprising Cas9 mRNA and sgRNAs.

FIGS. 17A, 17B, 17C, and 17D show in vivo results following administration of LNPs comprising Cas9 mRNA and sgRNAs. FIG. 17A shows percentage of total editing in liver. FIG. 17B shows the mean and standard deviation for the results of FIG. 17A. FIG. 17C shows serum TTR levels. FIG. 17D shows the mean and standard deviation for the results of FIG. 17B.

FIGS. 18A, 18B, and 18C show in vivo results following administration of LNPs comprising Cas9 mRNA and sgRNAs. FIG. 18A shows percentage of total editing in liver. FIG. 18B summarizes liver editing data. FIG. 18C shows serum TTR levels. MPK=milligrams per kilogram; BLOD=below level of detection.

FIGS. 19A, 19B, 19C, and 19D show interferon alpha (IFN-alpha, 19A), monocyte chemotactic protein 1 (MCP-1, 19B), interleukin 6 (IL-6, 19C), and tumor necrosis factor alpha (TNF-alpha, 19D) levels in serum after in vivo administration of LNPs comprising Cas9 mRNA and sgRNAs.

FIGS. 20A and 20B show editing in liver of FVII locus (FIG. 20A) and TTR locus (FIG. 20B) following in vivo administration of LNPs comprising Cas9 mRNA and sgRNAs.

FIGS. 21A, 21B, and 21C show schematics of an annotated sgRNA (SEQ ID NO: 341) (FIG. 21A), non-annotated dgRNA CR000686 (SEQ ID NO: 1) and TR000002 (SEQ ID NO: 188) (FIG. 21B), and annotated dgRNA CR000686 (SEQ ID NO: 1) and TR000002 (SEQ ID NO: 188) (FIG. 21C).

FIGS. 22A, 22B, and 22C show in vivo results following administration of LNPs comprising Cas9 mRNA and sgRNAs. FIG. 22A shows percentage of total editing of TTR locus in liver. FIG. 22B summarizes liver editing data. FIG. 22C shows serum TTR levels.

FIGS. 23A, 23B, and 23C show in vivo results following administration of LNPs comprising Cas9 mRNA and sgRNAs. FIG. 23A shows percentage of total editing of TTR locus in liver. FIG. 23B summarizes liver editing data. FIG. 23C shows serum TTR levels.

FIGS. 24A, 24B, and 24C show editing in primary mouse hepatocytes following administration of LNPs comprising Cas9 mRNA and sgRNAs. FIG. 24A shows editing percentage of total editing of TTR locus. FIG. 24B shows normalized transforms of editing percentage as a function of mRNA dose used to calculate EC50. FIG. 24C shows EC50 values for the LNPs tested.

DETAILED DESCRIPTION

Provided herein are modified guide RNAs, including dual and single guide RNAs for use in gene editing methods. The modified guides are more stable and show improved in vitro and in vivo efficacy as compared to their non-modified counterparts. Sequences of engineered and tested guide RNAs are shown in Table 4.

TABLE 4 SEQ ID NO Name Alias Description Sequence crRNA   1 CR000686 unmodified CCAGUCCAGCGAGGCAAAGGGUUUUAGAGCUAUGCUGUUUUG   2 CR003393 CR686-1 upper CCAGUCCAGCGAGGCAAAGGGUUUUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG   3 CR003394 CR686-2 partial upper CCAGUCCAGCGAGGCAAAGGGUUUUAGAGCUAmUmGmCmUmGmUmU mUmUmG   4 CR003395 CR686-3 partial upper CCAGUCCAGCGAGGCAAAGGGUUUUAGAGCUAUGCmUmGmUmUmUm UmG   5 CR003396 CR686-4 partial upper CCAGUCCAGCGAGGCAAAGGGUUUUAGAGCUAUGCUGUmUmUmUmG   6 CR003397 CR686-5 lower CCAGUCCAGCGAGGCAAAGGmGmUmUmUmUmAGAGCUAUGCUGUUU UG   7 CR003398 CR686-6 lower walk CCAGUCCAGCGAGGCAAAGGmGUUUUAGAGCUAUGCUGUUUUG   8 CR003399 CR686-7 lower walk CCAGUCCAGCGAGGCAAAGGGmUUUUAGAGCUAUGCUGUUUUG   9 CR003400 CR686-8 lower walk CCAGUCCAGCGAGGCAAAGGGUmUUUAGAGCUAUGCUGUUUUG  10 CR003401 CR686-9 lower walk CCAGUCCAGCGAGGCAAAGGGUUmUUAGAGCUAUGCUGUUUUG  11 CR003402 CR686-10 lower walk CCAGUCCAGCGAGGCAAAGGGUUUmUAGAGCUAUGCUGUUUUG  12 CR003403 CR686-11 lower walk CCAGUCCAGCGAGGCAAAGGGUUUUmAGAGCUAUGCUGUUUUG  13 CR003404 CR686-12 partial lower CCAGUCCAGCGAGGCAAAGGGmUmUmUmUAGAGCUAUGCUGUUUUG  14 CR003405 CR686-13 partial lower CCAGUCCAGCGAGGCAAAGGGUmUmUUAGAGCUAUGCUGUUUUG  15 CR003406 CR686-GC1 Lower GC CCAGUCCAGCGAGGCAAAGGGGCGCAGAGCUAUGCUGUUUUG  16 CR003407 CR686-GC3 Upper GC CCAGUCCAGCGAGGCAAAGGGUUUUAGAGCUAUGCUGGCGCG  17 CR003408 CR686-GC5 Lower Upper GC CCAGUCCAGCGAGGCAAAGGGGCGCAGAGCUAUGCUGGCGCG  18 CR003409 CR686 all mCmCmAmGmUmCmCmAmGmCmGmAmGmGmCmAmAmAmGmGm OMe GmUmUmUmUmAmGmAmGmCmUmAmUmGmCmUmGmUmUmUmUmG  19 CR003393- upper GUUUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only  20 CR003394- partial upper GUUUUAGAGCUAmUmGmCmUmGmUmUmUmUmG mod only  21 CR003395- partial upper GUUUUAGAGCUAUGCmUmGmUmUmUmUmG mod only  22 CR003396- partial upper GUUUUAGAGCUAUGCUGUmUmUmUmG mod only  23 CR003397- lower mGmUmUmUmUmAGAGCUAUGCUGUUUUG mod only  24 CR003398- lower walk mGUUUUAGAGCUAUGCUGUUUUG mod only  25 CR003399- lower walk GmUUUUAGAGCUAUGCUGUUUUG mod only  26 CR003400- lower walk GUmUUUAGAGCUAUGCUGUUUUG mod only  27 CR003401- lower walk GUUmUUAGAGCUAUGCUGUUUUG mod only  28 CR003402- lower walk GUUUmUAGAGCUAUGCUGUUUUG mod only  29 CR003403- lower walk GUUUUmAGAGCUAUGCUGUUUUG mod only  30 CR003404- partial lower GmUmUmUmUAGAGCUAUGCUGUUUUG mod only  31 CR003405- partial lower GUmUmUUAGAGCUAUGCUGUUUUG mod only  32 CR003721 CR686-14 upper and lower CCAGUCCAGCGAGGCAAAGGmGUUUmUmAGAmGmCmUmAmUmGmC mUmGmUmUmUmUmG  33  CR003722 CR686-15 lower combo CCAGUCCAGCGAGGCAAAGGmGUUUmUmAGAGCUAUGCUGUUUUG  34 CR003723 CR686-16 upper, lower CCAGUCCAGCGAGGCAAAGGmGUUUUmAGAmGmCmUmAmUmGmCm combo UmGmUmUmUmUmG  35 CR003724 CR686-17 lower combo CCAGUCCAGCGAGGCAAAGGmGUUUUmAGAGCUAUGCUGUUUUG  36 CR003725 CR686-18 upper, lower CCAGUCCAGCGAGGCAAAGGmGUUUUAGAmGmCmUmAmUmGmCmU combo mGmUmUmUmUmG  37 CR003726 CR686-19 nexus walk CCAGUCCAGCGAGGCAAAGGGUUUUAmGAmGmCmUmAmUmGmCmU mGmUmUmUmUmG  38 CR003727 CR686-20 nexus walk CCAGUCCAGCGAGGCAAAGGGUUUUAGmAmGmCmUmAmUmGmCmU mGmUmUmUmUmG  39 CR003728 CR686-21 nexus walk CCAGUCCAGCGAGGCAAAGGGUUUUAfGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  40 CR003729 CR686-22 nexus walk CCAGUCCAGCGAGGCAAAGGGUUUUAGfAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  41 CR003730 CR686-23 2′F lower walk CCAGUCCAGCGAGGCAAAGGGfUUUUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  42 CR003731 CR686-24 2′F lower walk CCAGUCCAGCGAGGCAAAGGGUfUUUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  43 CR003732 CR686-25 2′F lower walk CCAGUCCAGCGAGGCAAAGGGUUfUUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  44 CR003733 CR686-26 2′F lower walk CCAGUCCAGCGAGGCAAAGGGUUUfUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  45 CR003734 CR686-27 2′F lower combo CCAGUCCAGCGAGGCAAAGGfGfUfUfUfUfAGAmGmCmUmAmUmGmCm UmGmUmUmUmUmG  46 CR003735 CR686-28 lower alt CCAGUCCAGCGAGGCAAAGGfGmUfUmUfUmAGAmGmCmUmAmUmG mCmUmGmUmUmUmUmG  47 CR003736 CR686-29 lower alt CCAGUCCAGCGAGGCAAAGGmGfUmUfUmUfAGAmGmCmUmAmUmG mCmUmGmUmUmUmUmG  48 CR003737 CR686-GC6 Lower GC CCAGUCCAGCGAGGCAAAGGGUCUCAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  49 CR003738 CR686-GC7 Lower C walk CCAGUCCAGCGAGGCAAAGGGCUUUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  50 CR003739 CR686-GC8 Lower C walk CCAGUCCAGCGAGGCAAAGGGUCUUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  51 CR003740 CR686-GC9 Lower C walk CCAGUCCAGCGAGGCAAAGGGUUCUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  52 CR003741 CR686-GC10 Lower C walk CCAGUCCAGCGAGGCAAAGGGUUUCAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  53 CR003721- CR686-14- upper and lower mGUUUmUmAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  54 CR003722- CR686-15- lower combo mGUUUmUmAGAGCUAUGCUGUUUUG mod only mod only  55 CR003723- CR686-16- upper, lower mGUUUUmAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only combo  56 CR003724- CR686-17- lower combo mGUUUUmAGAGCUAUGCUGUUUUG mod only mod only  57 CR003725- CR686-18- upper, lower mGUUUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only combo  58 CR003726- CR686-19- nexus walk GUUUUAmGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  59 CR003727- CR686-20- nexus walk GUUUUAGmAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  60 CR003728- CR686-21- nexus walk GUUUUAfGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  61 CR003729- CR686-22- nexus walk GUUUUAGfAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  62 CR003730- CR686-23- 2′F lower walk GfUUUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  63 CR003731- CR686-24- 2′F lower walk GUfUUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  64 CR003732- CR686-25- 2′F lower walk GUUfUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  65 CR003733- CR686-26- 2′F lower walk GUUUfUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  66 CR003734- CR686-27- 2′F lower combo fGfUfUfUfUfAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  67 CR003735- CR686-28- lower alt fGmUfUmUfUmAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  68 CR003736- CR686-29- lower alt mGfUmUfUmUfAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  69 CR003737- CR686-GC6- Lower GC GUCUCAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  70 CR003738- CR686-GC7- Lower C walk GCUUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  71 CR003739- CR686-GC8- Lower C walk GUCUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  72 CR003740- CR686-GC9- Lower C walk GUUCUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  73 CR003741- CR686-GC10- Lower C walk GUUUCAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only  74 CR000705 unmodified UUACAGCCACGUCUACAGCAGUUUUAGAGCUAUGCUGUUUUG  75 CR004188 CR705-1 upper UUACAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  76  CR004189 CR705-2 partial upper UUACAGCCACGUCUACAGCAGUUUUAGAGCUAmUmGmCmUmGmUmU mUmUmG  77 CR004190 CR705-3 partial upper UUACAGCCACGUCUACAGCAGUUUUAGAGCUAUGCmUmGmUmUmUm UmG  78 CR004191 CR705-4 partial upper UUACAGCCACGUCUACAGCAGUUUUAGAGCUAUGCUGUmUmUmUmG  79 CR004192 CR705-5 lower UUACAGCCACGUCUACAGCAmGmUmUmUmUmAGAGCUAUGCUGUUU UG  80 CR004193 CR705-6 lower walk UUACAGCCACGUCUACAGCAmGUUUUAGAGCUAUGCUGUUUUG  81 CR004194 CR705-7 lower walk UUACAGCCACGUCUACAGCAGmUUUUAGAGCUAUGCUGUUUUG  82 CR004195 CR705-8 lower walk UUACAGCCACGUCUACAGCAGUmUUUAGAGCUAUGCUGUUUUG  83 CR004196 CR705-9 lower walk UUACAGCCACGUCUACAGCAGUUmUUAGAGCUAUGCUGUUUUG  84 CR004197 CR705-10 lower walk UUACAGCCACGUCUACAGCAGUUUmUAGAGCUAUGCUGUUUUG  85 CR004198 CR705-11 lower walk UUACAGCCACGUCUACAGCAGUUUUmAGAGCUAUGCUGUUUUG  86 CR004199 CR705-14 upper and lower UUACAGCCACGUCUACAGCAmGUUUmUmAGAmGmCmUmAmUmGmC mUmGmUmUmUmUmG  87 CR004200 CR705-15 lower combo UUACAGCCACGUCUACAGCAmGUUUmUmAGAGCUAUGCUGUUUUG  88 CR004201 CR705-16 upper, lower UUACAGCCACGUCUACAGCAmGUUUUmAGAmGmCmUmAmUmGmCm combo UmGmUmUmUmUmG  89 CR004202 CR705-17 lower combo UUACAGCCACGUCUACAGCAmGUUUUmAGAGCUAUGCUGUUUUG  90 CR004203 CR705-18 upper, lower UUACAGCCACGUCUACAGCAmGUUUUAGAmGmCmUmAmUmGmCmU combo mGmUmUmUmUmG  91 CR004204 CR705-19 nexus walk UUACAGCCACGUCUACAGCAGUUUUAmGAmGmCmUmAmUmGmCmU mGmUmUmUmUmG  92 CR004205 CR705-20 nexus walk UUACAGCCACGUCUACAGCAGUUUUAGmAmGmCmUmAmUmGmCmU mGmUmUmUmUmG  93 CR004206 CR705-21 nexus walk UUACAGCCACGUCUACAGCAGUUUUAfGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  94 CR004207 CR705-22 nexus walk UUACAGCCACGUCUACAGCAGUUUUAGfAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  95 CR004208 CR705-23 2′F lower walk UUACAGCCACGUCUACAGCAGfUUUUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  96 CR004209 CR705-24 2′F lower walk UUACAGCCACGUCUACAGCAGUfUUUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  97 CR004210 CR705-25 2′F lower walk UUACAGCCACGUCUACAGCAGUUfUUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  98 CR004211 CR705-26 2′F lower walk UUACAGCCACGUCUACAGCAGUUUfUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG  99 CR004212 CR705-27 2′F lower combo UUACAGCCACGUCUACAGCAfGfUfUfUfUfAGAmGmCmUmAmUmGmCm UmGmUmUmUmUmG 100 CR004213 CR705-28 lower alt UUACAGCCACGUCUACAGCAfGmUfUmUfUmAGAmGmCmUmAmUmG mCmUmGmUmUmUmUmG 101 CR004214 CR705-29 lower alt UUACAGCCACGUCUACAGCAmGfUmUfUmUfAGAmGmCmUmAmUmG mCmUmGmUmUmUmUmG 102 CR004215 CR705-GC1 Lower GC UUACAGCCACGUCUACAGCAGGCGCAGAGCUAUGCUGUUUUG 103 CR004216 CR705-GC3 Upper GC UUACAGCCACGUCUACAGCAGUUUUAGAGCUAUGCUGGCGCG 104 CR004188- CR705-1- upper GUUUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 105 CR004189- CR705-2- partial upper GUUUUAGAGCUAmUmGmCmUmGmUmUmUmUmG mod only mod only 106 CR004190- CR705-3- partial upper GUUUUAGAGCUAUGCmUmGmUmUmUmUmG mod only mod only 107 CR004191- CR705-4- partial upper GUUUUAGAGCUAUGCUGUmUmUmUmG mod only mod only 108 CR004192- CR705-5- lower mGmUmUmUmUmAGAGCUAUGCUGUUUUG mod only mod only 109 CR004193- CR705-6- lower walk mGUUUUAGAGCUAUGCUGUUUUG mod only mod only 110 CR004194- CR705-7- lower walk GmUUUUAGAGCUAUGCUGUUUUG mod only mod only 111 CR004195- CR705-8- lower walk GUmUUUAGAGCUAUGCUGUUUUG mod only- mod only mod only 112 CR004196- CR705-9- lower walk GUUmUUAGAGCUAUGCUGUUUUG mod only mod only 113 CR004197- CR705-10- lower walk GUUUmUAGAGCUAUGCUGUUUUG mod only mod only 114 CR004198- CR705-11- lower walk GUUUUmAGAGCUAUGCUGUUUUG mod only mod only 115 CR004199- CR705-14- upper and lower mGUUUmUmAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 116 CR004200- CR705-15- lower combo mGUUUmUmAGAGCUAUGCUGUUUUG mod only mod only 117 CR004201- CR705-16- upper, lower mGUUUUmAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only combo 118 CR004202- CR705-17- lower combo mGUUUUmAGAGCUAUGCUGUUUUG mod only mod only 119 CR004203- CR705-18- upper, lower mGUUUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only combo 120 CR004204- CR705-19- nexus walk GUUUUAmGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 121 CR004205- CR705-20- nexus walk GUUUUAGmAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 122 CR004206- CR705-21- nexus walk GAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 123 CR004207- CR705-22- nexus walk GfAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 124 CR004208- CR705-23- 2′F lower walk GfUUUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 125 CR004209- CR705-24- 2′F lower walk GUfUUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 126 CR004210- CR705-25- 2′F lower walk GUUfUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 127 CR004211- CR705-26- 2′F lower walk GUUUfUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 128 CR004212- CR705-27- 2′F lower combo fGfUfUfUfUfAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 129 CR004213- CR705-28- lower alt fGmUfUmUfUmAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 130 CR004214- CR705-29- lower alt mGfUmUfUmUfAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 131 CR000657 unmodified CAGGGCUCUUGAAGAUCUCCGUUUUAGAGCUAUGCUGUUUUG 132 CR004218 CR657-1 upper CAGGGCUCUUGAAGAUCUCCGUUUUAGAmGmCmUmAmUmGmCmUm GmUmUmUmUmG 133 CR004219 CR657-2 partial upper CAGGGCUCUUGAAGAUCUCCGUUUUAGAGCUAmUmGmCmUmGmUm UmUmUmG 134 CR004220 CR657-3 partial upper CAGGGCUCUUGAAGAUCUCCGUUUUAGAGCUAUGCmUmGmUmUmUm UmG 135 CR004221 CR657-4 partial upper CAGGGCUCUUGAAGAUCUCCGUUUUAGAGCUAUGCUGUmUmUmUmG 136 CR004222 CR657-5 lower CAGGGCUCUUGAAGAUCUCCmGmUmUmUmUmAGAGCUAUGCUGUUU UG 137 CR004223 CR657-6 lower walk CAGGGCUCUUGAAGAUCUCCmGUUUUAGAGCUAUGCUGUUUUG 138 CR004224 CR657-7 lower walk CAGGGCUCUUGAAGAUCUCCGmUUUUAGAGCUAUGCUGUUUUG 139 CR004225 CR657-8 lower walk CAGGGCUCUUGAAGAUCUCCGUmUUUAGAGCUAUGCUGUUUUG 140 CR004226 CR657-9 lower walk CAGGGCUCUUGAAGAUCUCCGUUmUUAGAGCUAUGCUGUUUUG 141 CR004227 CR657-10 lower walk CAGGGCUCUUGAAGAUCUCCGUUUmUAGAGCUAUGCUGUUUUG 142 CR004228 CR657-11 lower walk CAGGGCUCUUGAAGAUCUCCGUUUUmAGAGCUAUGCUGUUUUG 143 CR004229 CR657-14 upper and lower CAGGGCUCUUGAAGAUCUCCmGUUUmUmAGAmGmCmUmAmUmGmC mUmGmUmUmUmUmG 144 CR004230 CR657-15 lower combo CAGGGCUCUUGAAGAUCUCCmGUUUmUmAGAGCUAUGCUGUUUUG 145 CR004231 CR657-16 upper, lower CAGGGCUCUUGAAGAUCUCCmGUUUUmAGAmGmCmUmAmUmGmCm combo UmGmUmUmUmUmG 146 CR004232 CR657-17 lower combo CAGGGCUCUUGAAGAUCUCCmGUUUUmAGAGCUAUGCUGUUUUG 147 CR004233 CR657-18 upper, lower CAGGGCUCUUGAAGAUCUCCmGUUUUAGAmGmCmUmAmUmGmCmU combo mGmUmUmUmUmG 148 CR004234 CR657-19 nexus walk CAGGGCUCUUGAAGAUCUCCGUUUUAmGAmGmCmUmAmUmGmCmU mGmUmUmUmUmG 149 CR004235 CR657-20 nexus walk CAGGGCUCUUGAAGAUCUCCGUUUUAGmAmGmCmUmAmUmGmCmU mGmUmUmUmUmG 150 CR004236 CR657-21 nexus walk CAGGGCUCUUGAAGAUCUCCGUUUUAfGAmGmCmUmAmUmGmCmU mGmUmUmUmUmG 151 CR004237 CR657-22 nexus walk CAGGGCUCUUGAAGAUCUCCGUUUUAGfAmGmCmUmAmUmGmCmU mGmUmUmUmUmG 152 CR004238 CR657-23 2′F lower walk CAGGGCUCUUGAAGAUCUCCGfUUUUAGAmGmCmUmAmUmGmCmU mGmUmUmUmUmG 153 CR004239 CR657-24 2′F lower walk CAGGGCUCUUGAAGAUCUCCGUfUUUAGAmGmCmUmAmUmGmCmU mGmUmUmUmUmG 154 CR004240 CR657-25 2′F lower walk CAGGGCUCUUGAAGAUCUCCGUUfUUAGAmGmCmUmAmUmGmCmU mGmUmUmUmUmG 155 CR004241 CR657-26 2′F lower walk CAGGGCUCUUGAAGAUCUCCGUUUfUAGAmGmCmUmAmUmGmCmU mGmUmUmUmUmG 156 CR004242 CR657-27 2′F lower combo CAGGGCUCUUGAAGAUCUCCfGfUfUfUfUfAGAmGmCmUmAmUmGmC mUmGmUmUmUmUmG 157 CR004243 CR657-28 lower alt CAGGGCUCUUGAAGAUCUCCfGmUfUmUfUmAGAmGmCmUmAmUmG mCmUmGmUmUmUmUmG 158 CR004244 CR657-29 lower alt CAGGGCUCUUGAAGAUCUCCmGfUmUfUmUfAGAmGmCmUmAmUmG mCmUmGmUmUmUmUmG 159 CR004245 CR657-GC1 Lower GC CAGGGCUCUUGAAGAUCUCCGGCGCAGAGCUAUGCUGUUUUG 160 CR004246 CR657-GC3 Upper GC CAGGGCUCUUGAAGAUCUCCGUUUUAGAGCUAUGCUGGCGCG 161 CR004218- CR657-1- upper GUUUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 162 CR004219- CR657-2- partial upper GUUUUAGAGCUAmUmGmCmUmGmUmUmUmUmG mod only mod only 163 CR004220- CR657-3- partial upper GUUUUAGAGCUAUGCmUmGmUmUmUmUmG mod only mod only 164 CR004221- CR657-4- partial upper GUUUUAGAGCUAUGCUGUmUmUmUmG mod only mod only 165 CR004222- CR657-5- lower mGmUmUmUmUmAGAGCUAUGCUGUUUUG mod only mod only 166 CR004223- CR657-6- lower walk mGUUUUAGAGCUAUGCUGUUUUG mod only mod only 167 CR004224- CR657-7- lower walk GmUUUUAGAGCUAUGCUGUUUUG mod only mod only 168 CR004225- CR657-8- lower walk GUmUUUAGAGCUAUGCUGUUUUG mod only mod only 169 CR004226- CR657-9- lower walk GUUmUUAGAGCUAUGCUGUUUUG mod only mod only 170 CR004227- CR657-10- lower walk GUUUmUAGAGCUAUGCUGUUUUG mod only mod only 171 CR004228- CR657-11- lower walk GUUUUmAGAGCUAUGCUGUUUUG mod only mod only 172 CR004229- CR657-14- upper and lower mGUUUmUmAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 173 CR004230- CR657-15- lower combo mGUUUmUmAGAGCUAUGCUGUUUUG mod only mod only 174 CR004231- CR657-16- upper, lower mGUUUUmAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only combo 175 CR004232- CR657-17- lower combo mGUUUUmAGAGCUAUGCUGUUUUG mod only mod only 176 CR004233- CR657-18- upper, lower mGUUUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only combo 177 CR004234- CR657-19- nexus walk GUUUUAmGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 178 CR004235- CR657-20- nexus walk GUUUUAGmAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 179 CR004236- CR657-21- nexus walk GUUUUAfGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 180 CR004237- CR657-22- nexus walk GUUUUAGfAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 181 CR004238- CR657-23- 2′F lower walk GfUUUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 182 CR004239- CR657-24- 2′F lower walk GUfUUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 183 CR004240- CR657-25- 2′F lower walk GUUfUUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 184 CR004241- CR657-26- 2′F lower walk GUUUfUAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 185 CR004242- CR657-27- 2′F lower combo fGfUfUfUfUfAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 186 CR004243- CR657-28- lower alt fGmUfUmUfUmAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only 187 CR004244- CR657-29- lower alt mGfUmUfUmUfAGAmGmCmUmAmUmGmCmUmGmUmUmUmUmG mod only mod only trRNA 188 TR000002 unmodified AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG UGGCACCGAGUCGGUGCUUUUUUU 189 TR000110 TR2-v2-1 shortened tail AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG UGGCACCGAGUCGGUGCUUUU 190 TR000111 TR2-v2-2 Upper, hairpins mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUU AUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGm AmGmUmCmGmGmUmGmCmUmUmUmU 191 TR000112 TR2-v2-3 upper only mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU 192 TR000113 TR2-v2-4 hairpin 1 AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUm GmAmAmAmAmAmGmUmGGCACCGAGUCGGUGCUUUU 193 TR000114 TR2-v2-5 hairpin 2 AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG UGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU 194 TR000115 TR2-v2-6 upper, hairpin 2 mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGmGmCmAmCmCmGmAmGmUmCmGmGmUmG mCmUmUmUmU 195 TR000116 TR2-v2-7 both hairpins AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUm GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGm UmGmCmUmUmUmU 196 TR000117 TR2-v2-8 lower walk AACAGCAUAGCAAGUmUmAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGCUUUU 197 TR000118 TR2-v2-9 lower walk AACAGCAUAGCAAGUUAmAmAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGCUUUU 198 TR000119 TR2-v2-10 lower walk AACAGCAUAGCAAGUUAAAmAmUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGCUUUU 199 TR000120 TR2-v2-11 partial nexus AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGmUmUmAUCAACUUGA AAAAGUGGCACCGAGUCGGUGCUUUU 200 TR000121 TR2-v2-12 partial nexus AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAmUmCmAACUUGA AAAAGUGGCACCGAGUCGGUGCUUUU 201 TR000122 TR2-GC1 Lower GC AACAGCAUAGCAAGUUGCGCUAAGGCUAGUCCGUUAUCAACUUGAAAAAG UGGCACCGAGUCGGUGCUUUU 202 TR000123 TR2-GC3 upper GC GCCAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG UGGCACCGAGUCGGUGCUUUU 203 TR000124 TR2-GC5 Lower Upper GC GCCAGCAUAGCAAGUUGCGCUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGCUUUU 204 TR000125 TR2 all OMe mAmAmCmAmGmCmAmUmAmGmCmAmAmGmUmUmAmAmAmAmU mAmAmGmGmCmUmAmGmUmCmCmGmUmUmAmUmCmAmAmCmU mUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG mGmUmGmCmUmUmUmUmUmUmU 205 TR000126 TR2-v2-13 lower mAmAmCmAmGmCmAmUmAmGmCAAGUmUmAmAmAmAmUAAGGCU AGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmA mCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU 206 TR000127 TR2-v2-14 lower mAmAmCmAmGmCmAmUmAmGmCAAGUmUmAAAmAmUAAGGCUAG UCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmC mCmGmAmGmUmCmGmGmUmGmCmUmUmUmU 207 TR000128 TR2-v2-15 lower mAmAmCmAmGmCmAmUmAmGmCAAGUmUmAfAfAmAmUAAGGCUA GUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAm CmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU 208 TR000129 TR2-v2-16 lower alt mAmAmCmAmGmCmAmUmAmGmCAAGUmUfAmAfAmAfUAAGGCUAG UCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmC mCmGmAmGmUmCmGmGmUmGmCmUmUmUmU 209 TR000130 TR2-v2-17 lower alt mAmAmCmAmGmCmAmUmAmGmCAAGUfUmAfAmAfAmUAAGGCUAG UCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmC mCmGmAmGmUmCmGmGmUmGmCmUmUmUmU 210 TR000131 TR2-v2-18 nexus walk mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUU AUCmAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmG mAmGmUmCmGmGmUmGmCmUmUmUmU 211 TR000132 TR2-v2-19 nexus walk mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUU AUmCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmG mAmGmUmCmGmGmUmGmCmUmUmUmU 212 TR000133 TR2-v2-20 nexus walk mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUU AmUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmG mAmGmUmCmGmGmUmGmCmUmUmUmU 213 TR000134 TR2-v2-21 nexus walk mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUU mAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmG mAmGmUmCmGmGmUmGmCmUmUmUmU 214 TR000135 TR2-v2-22 nexus walk mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGU mUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCm GmAmGmUmCmGmGmUmGmCmUmUmUmU 215 TR000136 TR2-v2-23 nexus walk mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGm UUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCm GmAmGmUmCmGmGmUmGmCmUmUmUmU 216 TR000137 TR2-v2-24 nexus walk mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUU AUfCfAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmG mAmGmUmCmGmGmUmGmCmUmUmUmU 217 TR000138 TR2-v2-25 nexus walk mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUU fAfUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmG mAmGmUmCmGmGmUmGmCmUmUmUmU 218 TR000139 TR2-v2-26 nexus walk mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGfUf UAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmG mAmGmUmCmGmGmUmGmCmUmUmUmU 219 TR000140 TR2-v2-27 nexus walk mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAmGmGmCUAGUC CGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCm CmGmAmGmUmCmGmGmUmGmCmUmUmUmU 220 TR000141 TR2-v2-28 nexus walk mAmAmCmAmGmCmAmUmAmGmCAAGUUAAAAUAAGGCUAGUmCmC mGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCm CmGmAmGmUmCmGmGmUmGmCmUmUmUmU 221 TR000142 TR2-v2-29 bulge walk mAmAmCmAmGmCmAmUmAmGmCmAmAGUUAAAAUAAGGCUAGUCC GUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmC mGmAmGmUmCmGmGmUmGmCmUmUmUmU 222 TR000143 TR2-v2-30 bulge walk mAmAmCmAmGmCmAmUmAmGmCAAmGmUUAAAAUAAGGCUAGUCC GUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmC mGmAmGmUmCmGmGmUmGmCmUmUmUmU 223 TR000144 TR2-GC6 Lower GC AACAGCAUAGCAAGUUGAGAUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGCUUUU 224 TR000145 TR2-GC7 Lower GC walk AACAGCAUAGCAAGUUAAAGUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGCUUUU 225 TR000146 TR2-GC8 Lower GC walk AACAGCAUAGCAAGUUAAGAUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGCUUUU 226 TR000147 TR2-GC9 Lower GC walk AACAGCAUAGCAAGUUAGAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGCUUUU 227 TR000148 TR2-GC10 Lower GC walk AACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGCUUUU sgRNA 228 G000209 mC*mC*mA*GUCCAGCGAGGCAAAGGGUUUUAGAGCUAGAAAUAGCAAG UUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGCmU*mU*mU*U 229 G000262 G209-1 hairpin 2 mC*mC*mA*GUCCAGCGAGGCAAAGGGUUUUAGAGCUAGAAAUAGCAAG UUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUmGmGmCmAmC mCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 230 G000263 G209-2 hairpins mC*mC*mA*GUCCAGCGAGGCAAAGGGUUUUAGAGCUAGAAAUAGCAAG UUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU *mU 231 G000264 G209-3 tetraloop mC*mC*mA*GUCCAGCGAGGCAAAGGGUUUUAGAGCUAmGmAmAmAU AGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGCmU*mU*mU*U 232 G000265 G209-4 upper mC*mC*mA*GUCCAGCGAGGCAAAGGGUUUUAGAmGmCmUmAGAAAm UmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG GCACCGAGUCGGUGCmU*mU*mU*U 233 G000266 G209-5 upper and loop mC*mC*mA*GUCCAGCGAGGCAAAGGGUUUUAGAmGmCmUmAmGmA mAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAA AAAGUGGCACCGAGUCGGUGCmU*mU*mU*U 234 G000267 G209-6 upper, loop, mC*mC*mA*GUCCAGCGAGGCAAAGGGUUUUAGAmGmCmUmAmGmA hairpins mAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmU mUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG mGmUmGmCmU*mU*mU*mU 235 G000262- G209-1- hairpin 2 GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA mod only mod only CUUGAAAAAGUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCm U*mU*mU*mU 236 G000263- G209-2- hairpins GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAm mod only mod only AmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm UmCmGmGmUmGmCmU*mU*mU*mU 237 G000264- G209-3- tetraloop GUUUUAGAGCUAmGmAmAmAUAGCAAGUUAAAAUAAGGCUAGUCCGU mod only mod only UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU*mU*mU*U 238 G000265- G209-4- upper GUUUUAGAmGmCmUmAGAAAmUmAmGmCAAGUUAAAAUAAGGCUAG mod only mod only UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU*mU*mU*U 239 G000266- G209-5- upper and loop GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAG mod only mod only GCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU*mU* mU*U 240 G000267- G209-6- upper, loop, GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAG mod only mod only hairpins GCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGm CmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 241 G000211 end mod mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAGCUAGAAAUAGCAAG UUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGCmU*mU*mU*U 242 G000282 mod6 mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAm AmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUm UmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGm GmUmGmCmU*mU*mU*mU 243 G000201 unmod UUACAGCCACGUCUACAGCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAA UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU UU 244 G000331 G211-7 lower cr mU*mU*mA*CAGCCACGUCUACAGCAmGUUUUmAGAmGmCmUmAmG mAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmC mUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmC mGmGmUmGmCmU*mU*mU*mU 245 G000332 G211-8 lower cr mU*mU*mA*CAGCCACGUCUACAGCAfGfUfUfUfUfAGAmGmCmUmAmG mAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmC mUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmC mGmGmUmGmCmU*mU*mU*mU 246 G000333 G211-9 lower cr mU*mU*mA*CAGCCACGUCUACAGCAmGfUfUfUfUmAGAmGmCmUmAm GmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAm CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm CmGmGmUmGmCmU*mU*mU*mU 247 G000334 G211-10 lower tr mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAm AmAmUmAmGmCAAGUmUmAAAmAmUAAGGCUAGUCCGUUAUCAmAm CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm CmGmGmUmGmCmU*mU*mU*mU 248 G000335 G211-11 lower tr mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAm AmAmUmAmGmCAAGUmUmAfAfAmAmUAAGGCUAGUCCGUUAUCAmA mCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmU mCmGmGmUmGmCmU*mU*mU*mU 249 G000336 G211-12 lower tr mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAm AmAmUmAmGmCAAGUfUmAfAmAfAmUAAGGCUAGUCCGUUAUCAmA mCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmU mCmGmGmUmGmCmU*mU*mU*mU 250 G000337 G211-13 lower all mU*mU*mA*CAGCCACGUCUACAGCAmGUUUUmAGAmGmCmUmAmG mAmAmAmUmAmGmCAAGUmUmAAAmAmUAAGGCUAGUCCGUUAUCA mAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmG mUmCmGmGmUmGmCmU*mU*mU*mU 251 G000338 G211-14 lower all mU*mU*mA*CAGCCACGUCUACAGCAmGUUUUmAGAmGmCmUmAmG mAmAmAmUmAmGmCAAGUmUmAfAfAmAmUAAGGCUAGUCCGUUAUC AmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAm GmUmCmGmGmUmGmCmU*mU*mU*mU 252 G000339 G211-15 lower all mU*mU*mA*CAGCCACGUCUACAGCAmGUUUUmAGAmGmCmUmAmG mAmAmAmUmAmGmCAAGUfUmAfAmAfAmUAAGGCUAGUCCGUUAUC AmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAm GmUmCmGmGmUmGmCmU*mU*mU*mU 253 G000340 G211-16 lower all mU*mU*mA*CAGCCACGUCUACAGCAfGfUfUfUfUfAGAmGmCmUmAmG mAmAmAmUmAmGmCAAGUmUmAAAmAmUAAGGCUAGUCCGUUAUCA mAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmG mUmCmGmGmUmGmCmU*mU*mU*mU 254 G000341 G211-17 lower all mU*mU*mA*CAGCCACGUCUACAGCAfGfUfUfUfUfAGAmGmCmUmAmG mAmAmAmUmAmGmCAAGUmUmAfAfAmAmUAAGGCUAGUCCGUUAUC AmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAm GmUmCmGmGmUmGmCmU*mU*mU*mU 255 G000342 G211-18 lower all mU*mU*mA*CAGCCACGUCUACAGCAfGfUfUfUfUfAGAmGmCmUmAmG mAmAmAmUmAmGmCAAGUfUmAfAmAfAmUAAGGCUAGUCCGUUAUC AmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAm GmUmCmGmGmUmGmCmU*mU*mU*mU 256 G000343 G211-19 Bulge cr mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAmGmAmGmCmUmAmG mAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmC mUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmC mGmGmUmGmCmU*mU*mU*mU 257 G000344 G211-20 Bulge tr mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAm AmAmUmAmGmCmAmAmGmUUAAAAUAAGGCUAGUCCGUUAUCAmAm CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm CmGmGmUmGmCmU*mU*mU*mU 258 G000345 G211-21 nexus mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAm AmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUfAfUfCfAmAmCmU mUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG mGmUmGmCmU*mU*mU*mU 259 G000346 G211-22 nexus mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAm AmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAmUmCmAmAmC mUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmC mGmGmUmGmCmU*mU*mU*mU 260 G000347 G211-23 lower all mU*mU*mA*CAGCCACGUCUACAGCAfGfUfUfUfUfAmGmAmGmCmUmA mGmAmAmAmUmAmGmCmAmAmGmUmUmAfAfAmAmUAAGGCUAGU CCGUUAmUmCmAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCm AmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 261 G000348 G211-24 no PS mUmUmACAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmAm AmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUm GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGm UmGmCmUmUmUmU 262 G000349 G211-25 2 OMe PS mU*mU*ACAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmA mAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmU mGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmG mUmGmCmUmU*mU*mU 263 G000350 G211-26 2′F hairpin mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAm 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mGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 311 G000354- G208-9- lower cr mGfUfUfUfUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAA mod only mod only AUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUm GmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 312 G000355- G208-10- lower tr GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAAAmA mod only mod only mUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmU mGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 313 G000356- G208-11- lower tr GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAfAfAm mod only mod only AmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmU mGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 314 G000357- G208-12- lower tr GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUfUmAfAmAfA mod only mUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmU mGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 315 G000358- G208-13- lower all mGUUUUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAAA mod only mod only mAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGm UmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*m U 316 G000359- G208-14- lower all mGUUUUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAfAf mod only mod only AmAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU* mU 317 G000360- G208-15- lower all mGUUUUmAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUfUmAfAm mod only mod only AfAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU* mU 318 G000361- G208-16- lower all fGfUfUfUfUfAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAA mod only mod only AmAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmG mUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU* mU 319 G000362- G208-17- lower all fGfUfUfUfUfAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUmUmAfA mod only mod only fAmAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU *mU 320 G000363- G208-18- lower all fGfUfUfUfUfAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUfUmAfA mod only mod only mAfAmUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAm GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU *mU 321 G000364- G208-19- Bulge cr GUUUUAmGmAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAU mod only mod only AAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmG mGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 322 G000365- G208-20- Bulge tr GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCmAmAmGmUUAAA mod only mod only AUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUm GmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 323 G000366- G208-21- nexus GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAG mod only mod only GCUAGUCCGUUfAfUfCfAmAmCmUmUmGmAmAmAmAmAmGmUmGm GmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 324 G000367- G208-22- nexus GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAG mod only mod only GCUAGUCCGUUAmUmCmAmAmCmUmUmGmAmAmAmAmAmGmUmG mGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 325 G000368- G208-23- lower all fGfUfUfUfUfAmGmAmGmCmUmAmGmAmAmAmUmAmGmCmAmAmG mod only mod only mUmUmAfAfAmAmUAAGGCUAGUCCGUUAmUmCmAmAmCmUmUmG mAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmU mGmCmU*mU*mU*mU 326 G000369- G208-24- no PS GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAG mod only mod only GCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGm CmAmCmCmGmAmGmUmCmGmGmUmGmCmUmUmUmU 327 G000370- G208-25- 2 OMe PS GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAG mod only mod only GCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGm CmAmCmCmGmAmGmUmCmGmGmUmGmCmUmU*mU*mU 328 G000371- G208-26- 2′F hairpin GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAG mod only mod only GCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGm CmAmCmCmGmAmGfUfCfGfGfUfGfCfU*fU*fU*mU 329 G000372- G208-27- Alt hairpin GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAG mod only mod only GCUAGUCCGUUAUCAfAmCfUmUfGmAfAmAfAmAfGmUfGmGfCmAfCmC fGmAfGmUfCmGfGmUfGmCfU*mU*fU*mU 330 G000269 end mod mC*mC*mC*AUACUCCUACAGCACCAGUUUUAGAGCUAGAAAUAGCAAG UUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG GUGCmU*mU*mU*U 331 G000283 mod6 mC*mC*mC*AUACUCCUACAGCACCAGUUUUAGAmGmCmUmAmGmAm AmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUm UmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGm GmUmGmCmU*mU*mU*mU 332 G000285 unmod CCCAUACUCCUACAGCACCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU 342 G000537 G211-33 5′end 3xOMePS mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmA mAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmU mUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmGmCmUmUmUmU 343 G000538 G211-34 3′end 3xOMePS mUmUmACAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmA mAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUm UmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG mGmUmGmCmU*mU*mU*mU 344 G000539 G211-35 5xOMePS mU*mU*mA*mC*mA*GCCACGUCUACAGCAGUUUUAGAmGmCmUmA mGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAm AmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm UmCmGmGmUmG*mC*mU*mU*mU*mU 345 G000541 G211-37 3xOMePS+2PS mU*mU*mA*C*A*GCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGm AmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCm UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCm GmGmUmG*mC*mU*mU*mU*mU 346 G000542 G211-38 3xOMePS+7PS mU*mU*mA*C*A*G*C*C*A*C*GUCUACAGCAGUUUUAGAmGmCmUm AmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCA mAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmG mU*mC*mG*mG*mU*mG*mC*mU*mU*mU*mU 347 G000543 G211-39 invd abasic (invd)UUACAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmA mAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUm UmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmG mGmUmGmCmUmUmUmU(invd) 348 G000544 G211-40 invd abasic + (invd)mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAm 3xOMePS GmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmA mCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm UmCmGmGmUmGmCmU*mU*mU*mU(invd) 349 G000564 G211-42 3xMOE-PS moeU*moeU*moeA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmA mGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAm AmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm UmCmGmGmUmGmCmoeU*moeU*moeU*mU 350 G000545 G211-43 US loop PS mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmA*mG* mA*mA*mA*mUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAm AmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGm UmCmGmGmUmGmCmU*mU*mU*mU 351 G000546 G211-44 H1 loop PS mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmA mAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmU mU*mG*mA*mA*mA*mAmAmGmUmGmGmCmAmCmCmGmAmGmU mCmGmGmUmGmCmU*mU*mU*mU 352 G000547 G211-45 H2 loop PS mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmA mAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmU mUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmG*mA*mG*mU* mCmGmGmUmGmCmU*mU*mU*mU 353 G000548 G211-46 all loops PS mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmA*mG* mA*mA*mA*mUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAm AmCmUmU*mG*mA*mA*mA*mAmAmGmUmGmGmCmAmCmCmG* mA*mG*mU*mCmGmGmUmGmCmU*mU*mU*mU 354 Mod6 (with mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmG modifications not mAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmC shown in mUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm sequence listing) CmGmGmUmGmCmU*mU*mU*mU N = any nucleotide 355 Invariable region GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA only ACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU 356 Mod6 pattern; GUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAA invariable region GGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmG only mCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU 357 Variable and NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAA invariable region AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC UUUU 358 Mod6 with mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmG modifications mAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmC shown in mUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm sequence listing CmGmGmUmGmCmU*mU*mU*mU N = any nucleotide

“Guide RNA” and “gRNA” are used herein interchangeably to refer collectively to either an sgRNA, a trRNA (also known as tracrRNA), or a crRNA (also known as a CRISPR RNA). The crRNA and trRNA may be associated on one RNA molecule (single guide RNA [sgRNA]) or in two separate RNA molecules (dual guide RNA [dgRNA]). “Guide RNA” or “gRNA” refers to each type.

The trRNA sequences may be naturally-occurring, or the trRNA sequence may include modifications or variations compared to naturally-occurring sequences.

“Editing efficiency” or “editing percentage” or “percent editing” as used herein is the total number of sequence reads with insertions or deletions of nucleotides into the target region of interest over the total number of sequence reads following cleavage by a Cas RNP.

“Hairpin” as used herein describes a loop of nucleic acids that is created when a nucleic acid strand folds and forms base pairs with another section of the same strand. A hairpin may form a structure that comprises a loop or a U-shape. In some embodiments, a hairpin may be comprised of a RNA loop. Hairpins can be formed with two complementary sequences in a single nucleic acid molecule bind together, with a folding or wrinkling of the molecule. In some embodiments, hairpins comprise stem or stem loop structures.

“Regions” as used herein describes conserved groups of nucleic acids. Regions may also be referred to as “modules” or “domains.” Regions of a gRNA may perform particular functions, e.g., in directing endonuclease activity of the RNP, for example as described in Briner A E et al., Molecular Cell 56:333-339 (2014). Regions of a gRNA are described in Tables 1-3.

“Ribonucleoprotein” (RNP) or “RNP complex” as used herein describes a gRNA, for example, together with a nuclease, such as a Cas protein. In some embodiments, the RNP comprises Cas9 and gRNA.

“Stem loop” as used herein describes a secondary structure of nucleotides that form a base-paired “stem” that ends in a loop of unpaired nucleic acids. A stem may be formed when two regions of the same nucleic acid strand are at least partially complementary in sequence when read in opposite directions. “Loop” as used herein describes a region of nucleotides that do not base pair (i.e., are not complementary) that may cap a stem. A “tetraloop” describes a loop of 4 nucleotides. As used herein, the upper stem of a sgRNA may comprise a tetraloop.

In certain embodiments involving dgRNA, a “stem” region as used herein describes a secondary structure of nucleotides that forms a base-paired region between certain regions of a crRNA and trRNA (e.g., the lower and upper stem regions of each RNA). The “stem” region of a dgRNA may also be referred to in the art as a “flagpole” region.

“Treatment” as used herein covers any administration or application of a therapeutic for disease in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of one or more symptoms of the disease.

1. Types of Modifications

A. 2′-O-Methyl Modifications

Modified sugars are believed to control the puckering of nucleotide sugar rings, a physical property that influences oligonucleotide binding affinity for complementary strands, duplex formation, and interaction with nucleases. Substitutions on sugar rings can therefore alter the confirmation and puckering of these sugars. For example, 2′-O-methyl (2′-O-Me) modifications can increase binding affinity and nuclease stability of oligonucleotides, though as shown in the Examples, the effect of any modification at a given position in an oligonucleotide needs to be empirically determined.

The terms “mA,” “mC,” “mU,” or “mG” may be used to denote a nucleotide that has been modified with 2′-O-Me.

Modification of a ribonucleotide as 2′-O-methyl ribonucleotide can be depicted as follows:

B. 2′-O-(2-Methoxyethyl) Modifications

In some embodiments, the modification may be 2′-O-(2-methoxyethyl) (2′-O-moe). Modification of a ribonucleotide as a 2′-O-moe ribonucleotide can be depicted as follows:

The terms “moeA,” “moeC,” “moeU,” or “moeG” may be used to denote a nucleotide that has been modified with 2′-O-moe.

C. 2′-fluoro modifications

Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution. For example, 2′-fluoro (2′-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability.

In this application, the terms “fA,” “fC,” “fJ,” or “fG” may be used to denote a nucleotide that has been substituted with 2′-F.

Substitution of 2′-F can be depicted as follows:

D. Phosphorothioate modifications

Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one nonbridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S-oligos.

A “*” may be used to depict a PS modification. In this application, the terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3′) nucleotide with a PS bond.

In this application, the terms “mA*,” “mC*,” “mU*,” or “mG*” may be used to denote a nucleotide that has been substituted with 2′-O-Me and that is linked to the next (e.g., 3′) nucleotide with a PS bond. Similarly, the terms “fA*,” “fC*,” “fU*,” or “fG*” may be used to denote a nucleotide that has been substituted with 2′-F and that is linked to the next (e.g., 3′) nucleotide with a PS bond. Equivalents of a PS linkage or bond are encompassed by embodiments described herein.

The diagram below shows the substitution of S—into a nonbridging phosphate oxygen, generating a PS bond in lieu of a phosphodiester bond:

E. G-C substitutions

In some embodiments, gRNAs are modified with sequence substitutions that do not comprise chemical modifications. In some embodiments, modified gRNAs are engineered with G-C pairings (e.g., in lower and/or upper stem regions) that are not found in the parental gRNA sequence. In some embodiments, modified gRNAs are engineered with G-U mismatches (“GU wobbles” or mismatch pairings) that are not found in the parental gRNA sequence.

F. Inverted abasic modifications

Abasic nucleotides refer to those which lack nitrogenous bases. The figure below depicts an oligonucleotide with an abasic (also known as apurinic) site that lacks a base:

Inverted bases refer to those with linkages that are inverted from the normal 5′ to 3′ linkage (i.e., either a 5′ to 5′ linkage or a 3′ to 3′ linkage). For example:

An abasic nucleotide can be attached with an inverted linkage. For example, an abasic nucleotide may be attached to the terminal 5′ nucleotide via a 5′ to 5′ linkage, or an abasic nucleotide may be attached to the terminal 3′ nucleotide via a 3′ to 3′ linkage. An inverted abasic nucleotide at either the terminal 5′ or 3′ nucleotide may also be called an inverted abasic end cap. In this application, the terms “invd” indicates an inverted abasic nucleotide linkage.

The above modifications and their equivalents are included within the scope of the embodiments described herein.

2. Guide RNA Compositions

Compositions comprising guide RNA are encompassed. In some embodiments, the guide RNA comprises a trRNA. In some embodiments, the guide RNA comprises a crRNA. In some embodiments, the guide RNA comprises a crRNA and trRNA. In some embodiments, the guide RNA comprises a crRNA and trRNA on one RNA molecule as a sgRNA. In some embodiments, the guide RNA comprises a crRNA and trRNA on two RNA molecules as a dgRNA. In a dgRNA, the two RNA molecules may associate via base pairing.

In some embodiments, the guide RNA comprises a 5′ terminus region. In some embodiments, the guide RNA does not comprise a 5′ terminus region. In some embodiments, the 5′ terminus region comprises a “spacer” region as described in Briner A E et al., Molecular Cell 56: 333-339 (2014) for sgRNA (but applicable herein to all guide RNAs). In some embodiments, the 5′ terminus region comprises a 5′ end modification. A 5′ terminus region with or without a spacer region may be associated with a crRNA, trRNA, sgRNA and/or dgRNA The spacer region is also sometimes referred to herein, and by others, as a “guide region,” “guide domain” or “targeting domain.” A “target sequence” as used herein refers to a sequence of nucleic acid to which the guide region/domain directs a nuclease for cleavage. In some embodiments, a spyCas9 protein may be directed by a guide region/domain to a target sequence of a target nucleic acid molecule by the nucleotides present in the spacer region. In some embodiments, the guide RNA does not comprise a spacer region.

In some embodiments, the guide RNAs described herein comprise or consist of any of the sequences shown in Table 4. Note, however, that where a sequence shows a guide/spacer region, it should be recognized that the composition may comprise this region or not. Further, guide RNAs are encompassed that comprise the modifications of any of the sequences shown in Table 4, and identified therein by SEQ ID No. That is, the nucleotides may be the same or different, but the modification pattern shown may be the same or similar to a modification pattern of a guide sequence of Table 4. A modification pattern includes the relative position and identity of modifications of the gRNA or a region of the gRNA (e.g. 5′ terminus region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, 3′ terminus region). In some embodiments, the modification pattern contains at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the modifications of any one of the sequences shown in the sequence column of Table 4, or over one or more regions of the sequence. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to the modification pattern of any one of the sequences shown in the sequence column of Table 4. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical over one or more regions of the sequence shown in Table 4, e.g., a 5′ terminus region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, and/or 3′ terminus region. For example, in some embodiments, a guide RNA is encompassed wherein the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical to the modification pattern of a sequence over the 5′ terminus region. In some embodiments, a guide RNA is encompassed wherein the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical over the lower stem. In some embodiments, a guide RNA is encompassed wherein the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical over the bulge. In some embodiments, a guide RNA is encompassed wherein the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical over the upper stem. In some embodiments, a guide RNA is encompassed wherein the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical over the nexus. In some embodiments, a guide RNA is encompassed wherein the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical over the hairpin 1. In some embodiments, a guide RNA is encompassed wherein the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical over the hairpin 2. In some embodiments, a guide RNA is encompassed wherein the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identical over the 3′ terminus. In some embodiments, the modification pattern differs from the modification pattern of a sequence of Table 4, or a region (e.g. 5′ terminus, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3′ terminus) of such a sequence, at 0, 1, 2, 3, 4, 5, or 6 nucleotides. In some embodiments, the gRNA comprises modifications that differ from the modifications of a sequence of Table 4, at 0, 1, 2, 3, 4, 5, or 6 nucleotides. In some embodiments, the gRNA comprises modifications that differ from modifications of a region (e.g. 5′ terminus, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3′ terminus) of a sequence of Table 4, at 0, 1, 2, 3, 4, 5, or 6 nucleotides.

In some embodiments, the gRNA comprises a 2′-O-methyl (2′-O-Me) modified nucleotide. In some embodiments, the gRNA comprises a 2′-O-(2-methoxyethyl) (2′-O-moe) modified nucleotide. In some embodiments, the gRNA comprises a 2′-fluoro (2′-F) modified nucleotide. In some embodiments, the gRNA comprises a phosphorothioate (PS) bond between nucleotides.

In some embodiments, the gRNA comprises a 5′ end modification, a 3′ end modification, or 5′ and 3′ end modifications. In some embodiments, the 5′ end modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the 5′ end modification comprises a 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxyethyl) (2′-O-moe), and/or 2′-fluoro (2′-F) modified nucleotide. In some embodiments, the 5′ end modification comprises at least one phosphorothioate (PS) bond and one or more of a 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxyethyl) (2′-O-moe), and/or 2′-fluoro (2′-F) modified nucleotide. The end modification may comprise a phosphorothioate (PS), 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxyethyl) (2′-O-moe), and/or 2′-fluoro (2′-F) modification. Equivalent end modifications are also encompassed by embodiments described herein. In some embodiments, the gRNA comprises an end modification in combination with a modification of one or more regions of the gRNA.

A. Compositions of sgRNAs

In some embodiments, the compositions and methods of the invention comprise gRNA comprising a crRNA and trRNA that direct a nuclease such as Cas9 to a target DNA sequence. In some embodiments, the gRNAs described herein may be associated on one RNA molecule (single guide RNA or sgRNA).

In some embodiments, the invention comprises a sgRNA comprising or consisting of any one of the sequences described in SEQ ID Nos: 228-332.

In some embodiments, a sgRNA comprising any one of the modified sequences of SEQ ID Nos: 235-240, 265-285, and 309-329 is provided. In some embodiments, a sgRNA comprising any one of the modified sequences of SEQ ID Nos: 235-240, 265-285, and 309-329, wherein the sgRNA further comprises a 5′ “spacer” sequence (“guide sequence”) that is complementary to a target sequence, and directs a Cas9 to its target for cleavage is encompassed. In some instances, the invention comprises sgRNA comprising nucleic acids having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to the nucleic acids of any one of SEQ ID Nos: 235-240, 265-285, and 309-329, wherein the modification pattern is identical to the modification pattern shown in the reference sequence identifier.

1. Domains of sgRNAs

Briner A E et al., Molecular Cell 56:333-339 (2014) describes functional domains of sgRNAs, referred to herein as “domains”, including the “spacer” domain responsible for targeting, the “lower stem”, the “bulge”, “upper stem” (which may include a tetraloop), the “nexus”, and the “hairpin 1” and “hairpin 2” domains. See, Briner et al. at page 334, FIG. 1A.

Table 1 and FIG. 21A provide a description of the domains of a sgRNA as used herein. In Table 1, the “n” between regions represents a variable number of nucleotides, for example, from 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. In some embodiments, n equals 0. In some embodiments, n equals 1.

TABLE 1 Regions of sgRNA (linear view, 5′ to 3′) LS1-6 B1-2 US1-12 B3-6 5′ terminus (n) lower stem n bulge n upper stem n bulge n H1-1 thru H2-1 thru LS7-12 N1-18 H1-12 H2-15 lower stem n nexus n hairpin 1 n hairpin 2 3′ terminus

a) 5′ terminus region

In some embodiments, the sgRNA comprises nucleotides at the 5′ terminus as shown in Table 1. In some embodiments, the 5′ terminus of the sgRNA comprises a spacer or guide region that functions to direct a Cas protein to a target nucleotide sequence. In some embodiments, the 5′ terminus does not comprise a spacer or guide region. In some embodiments, the 5′ terminus comprises a spacer and additional nucleotides that do not function to direct a Cas protein to a target nucleotide region.

In some embodiments, the guide region comprises the first 1-10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides at the 5′ end of the sgRNA. In some embodiments, the guide region comprises 20 nucleotides. In some embodiments, the guide region may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more nucleotides. In some embodiments, the guide region may comprise 17 nucleotides. In some embodiments, the guide region may comprise 18 nucleotides. In some embodiments, the guide region may comprise 19 nucleotides.

In some embodiments, the selection of the guide region is determined based on target sequences within the gene of interest for editing. For example, in some embodiments, the sgRNA comprises a guide region that is complementary to target sequences of a gene of interest.

In some embodiments, the target sequence in the gene of interest may be complementary to the guide region of the sgRNA. In some embodiments, the degree of complementarity or identity between a guide region of a sgRNA and its corresponding target sequence in the gene of interest may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide region of a sgRNA and the target region of a gene of interest may be 100% complementary or identical. In other embodiments, the guide region of a sgRNA and the target region of a gene of interest may contain at least one mismatch. For example, the guide region of a sgRNA and the target sequence of a gene of interest may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches, where the total length of the target sequence is at least about 17, 18, 19, 20 or more base pairs. In some embodiments, the guide region of a sgRNA and the target region of a gene of interest may contain 1-6 mismatches where the guide sequence comprises at least about 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide region of a sgRNA and the target region of a gene of interest may contain 1, 2, 3, 4, 5, or 6 mismatches where the guide sequence comprises about 20 nucleotides. The 5′ terminus may comprise nucleotides that are not considered guide regions (i.e., do not function to direct a cas9 protein to a target nucleic acid).

b) Lower stem

In some embodiments, the sgRNA comprises a lower stem (LS) region that when viewed linearly, is separated by a bulge and upper stem regions. See Table 1.

In some embodiments, the lower stem regions comprise 1-12 nucleotides, e.g. in one embodiment the lower stem regions comprise LS1-LS12. In some embodiments, the lower stem region comprises fewer nucleotides than shown in Table 1 and FIG. 21A. In some embodiments, the lower stem region comprises more nucleotides than shown in Table 1 and FIG. 21A. When the lower stem region comprises fewer or more nucleotides than shown in the schematic of Table 1 and FIG. 21A, the modification pattern, as will be apparent to the skilled artisan, should be maintained.

In some embodiments, the lower stem region has nucleotides that are complementary in nucleic acid sequence when read in opposite directions. In some embodiments, the complementarity in nucleic acid sequence of lower stem leads to a secondary structure of a stem in the sgRNA (e.g., the regions may base pair with one another). In some embodiments, the lower stem regions may not be perfectly complimentary to each other when read in opposite directions.

c) Bulge

In some embodiments, the sgRNA comprises a bulge region comprising six nucleotides, B1-B6. When viewed linearly, the bulge region is separated into two regions. See Table 1. In some embodiments, the bulge region comprises six nucleotides, wherein the first two nucleotides are followed by an upper stem region, followed by the last four nucleotides of the bulge. In some embodiments, the bulge region comprises fewer nucleotides than shown in Table 1 and FIG. 21A. In some embodiments, the bulge region comprises more nucleotides than shown in Table 1 and FIG. 21A. When the bulge region comprises fewer or more nucleotides than shown in the schematic of Table 1 and FIG. 21A, the modification pattern, as will be apparent to the skilled artisan, should be maintained.

In some embodiments, the presence of a bulge results in a directional kink between the upper and lower stem modules in a sgRNA.

d) Upper stem

In some embodiments, the sgRNA comprises an upper stem region comprising 12 nucleotides. In some embodiments, the upper stem region comprises a loop sequence. In some instances, the loop is a tetraloop (loop consisting of four nucleotides).

In some embodiments, the upper stem region comprises fewer nucleotides than shown in Table 1 and FIG. 21A. In some embodiments, the upper stem region comprises more nucleotides than shown in Table 1 and FIG. 21A. When the upper stem region comprises fewer or more nucleotides than shown in the schematic of Table 1 and FIG. 21A, the modification pattern, as will be apparent to the skilled artisan, should be maintained.

In some embodiments, the upper stem region has nucleotides that are complementary in nucleic acid sequence when read in opposite directions. In some embodiments, the complementarity in nucleic acid sequence of upper stem leads to a secondary structure of a stem in the sgRNA (e.g., the regions may base pair with one another). In some embodiments, the upper stem regions may not be perfectly complimentary to each other when read in opposite directions.

e) Nexus

In some embodiments, the sgRNA comprises a nexus region that is located between the lower stem region and the hairpin 1 region. In some embodiments, the nexus comprises 18 nucleotides. In some embodiments, the nexus region comprises nucleotides N1 through N18 as shown in Table 1 and FIG. 21A.

In some embodiments, the nexus region comprises fewer nucleotides than shown in Table 1 and FIG. 21A. In some embodiments, the nexus region comprises more nucleotides than shown in Table 1 and FIG. 21A. When the nexus region comprises fewer or more nucleotides than shown in the schematic of Table 1 and FIG. 21A, the modification pattern, as will be apparent to the skilled artisan, should be maintained.

In some embodiments, the nexus region has nucleotides that are complementary in nucleic acid sequence when read in opposite directions. In some embodiments, the complementarity in nucleic acid sequence leads to a secondary structure of a stem and/or stem loop in the sgRNA (e.g., certain nucleotides in the nexus region may base pair with one another). In some embodiments, the nexus regions may not be perfectly complimentary to each other when read in opposite directions.

f) Hairpin

In some embodiments, the sgRNA comprises one or more hairpin regions. In some embodiments, the hairpin region is downstream of (e.g., 3′ to) the nexus region. In some embodiments, the region of nucleotides immediately downstream of the nexus region is termed “hairpin 1” or “H1”. In some embodiments, the region of nucleotides 3′ to hairpin 1 is termed “hairpin 2” or “H2”. In some embodiments, the hairpin region comprises hairpin 1 and hairpin 2. In some embodiments, the sgRNA comprises only hairpin 1 or hairpin 2.

In some embodiments, the hairpin 1 region comprises 12 nucleic acids immediately downstream of the nexus region. In some embodiments, the hairpin 1 region comprises nucleotides H1-1 through H1-12 as shown in Table 1 and FIG. 21A.

In some embodiments, the hairpin 2 region comprises 15 nucleic acids downstream of the hairpin 1 region. In some embodiments, the hairpin 2 region comprises nucleotides H2-1 through H2-15 as shown in Table 1 and FIG. 21A.

In some embodiments, one or more nucleotides is present between the hairpin 1 and the hairpin 2 regions. The one or more nucleotides between the hairpin 1 and hairpin 2 region may be modified or unmodified. In some embodiments, hairpin 1 and hairpin 2 are separated by one nucleotide. In some embodiments, the hairpin regions comprise fewer nucleotides than shown in Table 1 and FIG. 21A. In some embodiments, the hairpin regions comprise more nucleotides than shown in Table 1 and FIG. 21A. When a hairpin region comprises fewer or more nucleotides than shown in the schematic of Table 1 and FIG. 21A, the modification pattern, as will be apparent to the skilled artisan, should be maintained.

In some embodiments, a hairpin region has nucleotides that are complementary in nucleic acid sequence when read in opposite directions. In some embodiments, the hairpin regions may not be perfectly complimentary to each other when read in opposite directions (e.g., the top or loop of the hairpin comprises unpaired nucleotides).

In some embodiments, the sgRNA comprises replacement of hairpin 1 with nucleotides “n”, wherein “n” is an integer between 1 and 50, 40, 30, 20, 15, 10, 5, 4, 3, and 2. In some embodiments, the hairpin 1 region of a sgRNA is replaced by 2 nucleotides.

g) 3′ Terminus Region

In some embodiments, the sgRNA comprises nucleotides after the hairpin region(s). In some embodiments, the 3′ terminus region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 or more nucleotides, e.g. that are not associated with the secondary structure of a hairpin. In some embodiments, the 3′ terminus region comprises 1, 2, 3, or 4 nucleotides that are not associated with the secondary structure of a hairpin. In some embodiments, the 3′ terminus region comprises 4 nucleotides that are not associated with the secondary structure of a hairpin. In some embodiments, the 3′ terminus region comprises 1, 2, or 3 nucleotides that are not associated with the secondary structure of a hairpin.

2. Modifications of sgRNAs

In some embodiments, the invention comprises a sgRNA comprising one or more modifications within one or more of the following regions: the nucleotides at the 5′ terminus; the lower stem region; the bulge region; the upper stem region; the nexus region; the hairpin 1 region; the hairpin 2 region; and the nucleotides at the 3′ terminus.

In some embodiments, the modification comprises a 2′-O-methyl (2′-O-Me) modified nucleotide. In some embodiments, the modification comprises a 2′-O-(2-methoxyethyl) (2′-O-moe) modified nucleotide. In some embodiments, the modification comprises a 2′-fluoro (2′-F) modified nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) bond between nucleotides.

In some embodiments, the sgRNA comprises modifications at 1, 2, 3, or 4 of the first 4 nucleotides at its 5′ end. In some embodiments, the first three or four nucleotides at the 5′ terminus, and the last three or four nucleotides at the 3′ terminus are modified. In some embodiments, the first four nucleotides at the 5′ terminus, and the last four nucleotides at the 3′ terminus are linked with phosphorothioate (PS) bonds. In some embodiments, the modification comprises 2′-O-Me. In some embodiments, the modification comprises 2′-F. In some embodiments, the modification comprises 2′-O-moe.

In some embodiments, the sgRNA comprises modifications at 1, 2, 3, or 4 of the first 4 nucleotides at the 5′ end. In some embodiments, the sgRNA comprises modifications at 1, 2, 3, or 4 of the first 4 nucleotides at the 3′ end. In some embodiments, the first four nucleotides at the 5′ terminus and the last four nucleotides at the 3′ terminus are linked with a PS bond, and the first three nucleotides at the 5′ terminus and the last three nucleotides at the 3′ terminus comprise 2′-O-Me or 2′-O-moe modifications.

In some embodiments, the first four nucleotides at the 5′ terminus and the last four nucleotides at the 3′ terminus are linked with a PS bond, and the first three nucleotides at the 5′ terminus and the last three nucleotides at the 3′ terminus comprise 2′-F modifications.

In some embodiments, a sgRNA is provided wherein LS1, LS6, LS7, LS8, LS11, and LS12 are modified with 2′-O-Me. In some embodiments, each of the nucleotides in the bulge region of the sgRNA are modified with 2′-O-Me. In some embodiments, each of the nucleotides in the upper stem region of the sgRNA are modified with 2′-O-Me. In some embodiments, N16, N17, and N18 in the nexus region of the sgRNA are modified with 2′-O-Me. In some embodiments, each of the nucleotides in the hairpin 1 region of the sgRNA are modified with 2′-O-Me. In some embodiments, each of the nucleotides in the hairpin 2 region of the sgRNA are modified with 2′-O-Me.

In some embodiments, the sgRNA comprises 2′-O-Me modified nucleotides at the following nucleotides: the first three nucleotides at the 5′ terminus; LS1, LS6, LS7, LS8, LS11, and LS12; B1 and B2 in the bulge region; each of the nucleotides in the upper stem region of the sgRNA; N16, N17, and N18 in the nexus region; each of the nucleotides in the hairpin 1 region; each of the nucleotides in the hairpin 2 region; and last four nucleotides at the 3′ terminus.

In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus. In some embodiments, the sgRNA further comprises 2′-O-Me or 2′-F modified nucleic acids at the first three nucleotides at the 5′ terminus, and 2′-O-Me or 2′-F modified nucleic acids at the last four nucleotides at the 3′ terminus. In some embodiments, LS9 and LS10 are modified with 2′-F. In some embodiments, N15, N16, N17, and N18 are modified with 2′-F. In some embodiments, H2-9, H2-10, H2-11, H2-12, H2-13, HS-14, and H2-15 are modified with 2′-F. In some embodiments, the second to last, third to last, and fourth to last nucleotides at the 3′ terminus are modified with 2′-F

In some embodiments, a single guide RNA (sgRNA) is provided comprising 2′-F modified nucleic acids at the following nucleotides: LS9 and LS10 in the lower stem region; N15, N16, N17, and N18 in the nexus region; and H2-9, H2-10, H2-11, H2-12, H2-13, HS-14, and H2-15 in the hairpin 2 region. In some embodiments, the sgRNA further comprises 2′-F modified nucleotides at the second to last, third to last, and fourth to last nucleotides at the 3′ terminus. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus. In some embodiments, the sgRNA further comprises 2′-O-Me or 2′-F modified nucleic acids at the first three nucleotides at the 5′ terminus, and 2′-O-Me or 2′-F modified nucleic acids at three of the last four nucleotides at the 3′ terminus.

In some embodiments, a single guide RNA (sgRNA) is provided comprising 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ terminus; 2′-O-Me modified nucleotides at LS1 and LS6; 2′-O-Me modified nucleotides at US1-US12; 2′-O-Me modified nucleotides at H1-1-H1-12; a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin 2; 2′-O-Me modified nucleotides at H2-1-H2-15; and 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ terminus. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

In some embodiments, a single guide RNA (sgRNA) is provided comprising 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ terminus; 2′-F modified nucleotides at LS1-LS6; 2′-O-Me modified nucleotides at US1-US12; 2′-O-Me modified nucleotides at H1-1-H1-12; a 2′-O-Me modified nucleotide at “n” between Hairpin 1 and Hairpin 2; 2′-O-Me modified nucleotides at H2-1-H2-15; and 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ terminus. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

In some embodiments, a single guide RNA (sgRNA) is provided comprising 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ terminus; 2′-F modified nucleotides at LS2-LS5; 2′-O-Me modified nucleotides at LS1 and LS6; 2′-O-Me modified nucleotides at US1-US12; 2′-O-Me modified nucleotides at H1-1-H1-12; a 2′-O-Me modified nucleotide at “n” between Hairpin 1 and Hairpin 2; 2′-O-Me modified nucleotides at H2-1-H2-15; and 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ terminus. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

In some embodiments, a single guide RNA (sgRNA) is provided comprising 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ terminus; 2′-O-Me modified nucleotides at US1-US12; 2′-O-Me modified nucleotides at LS7, LS8, LS11, and LS12; 2′-O-Me modified nucleotides at H1-1-H1-12; a 2′-O-Me modified nucleotide at “n” between Hairpin 1 and Hairpin 2; 2′-O-Me modified nucleotides at H2-1-H2-15; and 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ terminus. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

In some embodiments, a single guide RNA (sgRNA) is provided comprising 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ terminus; 2′-O-Me modified nucleotides at US1-US12; 2′-O-Me modified nucleotides at LS8, LS10, and LS12; 2′-O-F modified nucleotides at LS7, LS9, and LS11; 2′-O-Me modified nucleotides at H1-1-H1-12; a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin 2; 2′-O-Me modified nucleotides at H2-1-H2-15; and 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ terminus. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

In some embodiments, a single guide RNA (sgRNA) is provided comprising 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ terminus; 2′-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11, and LS12; 2′-O-Me modified nucleotides at US1-US12; 2′-O-Me modified nucleotides at H1-1-H1-12; a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin 2; 2′-O-Me modified nucleotides at H2-1-H2-15; and 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ terminus. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

In some embodiments, a single guide RNA (sgRNA) is provided comprising 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ terminus; 2′-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11, and LS12; 2′-F modified nucleotides at LS9 and LS10; 2′-O-Me modified nucleotides at US1-US12; 2′-O-Me modified nucleotides at H1-1-H1-12; a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin 2; 2′-O-Me modified nucleotides at H2-1-H2-15; and 2′-O-Me modified nucleotides at the last four nucleotides at the 3′ terminus. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

In some embodiments, a single guide RNA (sgRNA) is provided comprising 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ terminus; 2′-O-Me modified nucleotides at US1-US12; 2′-O-Me modified nucleotides at H1-1-H1-12; a 2′-O-Me modified nucleotide between Hairpin 1 and Hairpin 2; 2′-O-Me modified nucleotides at H2-1-H2-8; 2′-F modified nucleotides at H2-9-H2-15; 2′-F modified nucleotides at the second from last, third from last, and fourth from last nucleotide at the 3′ terminus; and a 2′-O-Me modified nucleotide at the last nucleotide at the 3′ terminus. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

In some embodiments, a single guide RNA (sgRNA) is provided comprising 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ terminus; 2′-O-Me modified nucleotides at US1-US12; 2′-O-Me modified nucleotides at H1-2, H1-4, H1-6, H1-8, H1-10, and H1-12; 2′-F modified nucleotides at H1-1, H1-3, H1-5, H1-7, H1-9, and H1-11; a 2′-F modified nucleotide between Hairpin 1 and Hairpin 2; 2′-F modified nucleotides at H2-2, H2-4, H2-6, H2-8, H2-10, H2-12; and H2-14; 2′-O-Me modified nucleotides at H2-1, H2-3, H2-5, H2-7, H2-9, H2-11; H2-13, and H2-15; 2′-F modified nucleotides at the second from last, and fourth from last nucleotide at the 3′ terminus; and 2′-O-Me modified nucleotide at the third from last, and last nucleotide at the 3′ terminus. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

Disclosed herein, in some embodiments, is a single guide RNA (sgRNA) comprising 2′-O-Me modifications at nucleotides LS8, LS10, LS12, H1-2, H1-4, H1-6, H1-8, H1-10, H1-12, H2-1, H2-3, H2-5, H2-7, H2-9, H2-11, H2-13, and H2-15; and 2′-F modifications at LS7, LS9, LS11; H1-1, H1-3, H1-5, H1-7, H1-9, H1-11, H1-13, H2-2, H2-4, H2-6, H2-8, H2-10, H2-12, and H2-14. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus. In some embodiments, the sgRNA further comprises 2′-O-Me modified nucleotides at the last and third to last nucleotide at the 3′ terminus; and 2′-F modified nucleotides at the second to last and third to last nucleotide at the 3′ terminus.

Disclosed herein, in some embodiments, is a sgRNA comprising the nucleic acids of any one of SEQ ID Nos: 228-232. Disclosed herein, in some embodiments, is a sgRNA comprising the nucleic acids of any one of SEQ ID Nos: 235-240, 265-285, and 309-329. Disclosed herein, in some embodiments, is a sgRNA comprises nucleic acids having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to the nucleic acids of any one of SEQ ID Nos: 235-240, 265-285, and 309-329, wherein the modification pattern is identical to the modification pattern shown in the reference sequence identifier. In some embodiments, the sgRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

In some embodiments, a sgRNA comprising a 5′ end modification and one or more modifications in one or more of: the upper stem region; the hairpin 1 region; and the hairpin 2 region is provided, wherein the 5′ end modification comprises at least two phosphorothioate linkages within the first seven nucleotides of the 5′ terminus.

In some embodiments, a sgRNA comprising a 5′ end modification and one or more modifications in one or more of: the upper stem region; the hairpin 1 region; and the hairpin 2 region is provided, wherein the 5′ end modification comprises one or more phosphorothioate linkages at the 5′ end of the RNA. In some embodiments, one or more phorphorothioate bonds link the 5′ terminal nucleotides.

In some embodiments, a sgRNA comprising a 5′ end modification and one or more modifications in one or more of: the upper stem region; the hairpin 1 region; and the hairpin 2 region is provided, wherein the 5′ end modification comprises one or more phosphorothioate linkages within the first seven nucleotides of the 5′ terminus.

In some embodiments, a sgRNA comprising any one of the modified sgRNA sequences of SEQ ID Nos: 228-332 is provided.

In some embodiments, a sgRNA comprising or consisting of any one of the modified sgRNA sequences of SEQ ID Nos: 235-240, 265-285, and 309-329 is provided.

In some embodiments, the invention comprises a sgRNA comprising any one of the modified sequences of SEQ ID Nos: 235-240, 265-285, and 309-329, wherein the sgRNA further comprises a 5′ spacer sequence that is at least partially complementary to a target sequence, and directs a Cas9 to its target for cleavage.

In some embodiments, the invention comprises a sgRNA comprising nucleotides having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to the nucleotides of any one of SEQ ID Nos: 235-240, 265-285, and 309-329, wherein the modification pattern is identical to the modification pattern shown in the reference sequence identifier. That is, the nucleotides A, U, C, and G may differ by 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% compared to what is shown in in the sequences, but the modification remains unchanged.

In some embodiments, the invention comprises a sgRNA comprising one or more modifications within one or more of the following regions: the nucleotides at the 5′ terminus; the lower stem region; the bulge region; the upper stem region; the; the nexus region; the hairpin 1 region; the hairpin 2 region; and the nucleotides at the 3′ terminus.

In some embodiments, the modification comprises a 2′-O-methyl (2′-O-Me) modified nucleotide. In some embodiments, the modification comprises a 2′-fluoro (2′-F) modified nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the modification comprises an inverted abasic nucleotide.

In some embodiments, a sgRNA is provided comprising 2′-O-Me modified nucleotides at: the first three nucleotides in the 5′ terminus; LS1, LS6, LS7, LS8, LS11, and LS12 in the lower stem; B1 and B2 in the bulge region; each of the nucleotides in the upper stem region; N16, N17, and N18 in the nexus region; each of the nucleotides in the hairpin 1 region; one nucleotide between hairpin 1 and hairpin 2; each of the nucleotides in the hairpin 2 region; and the last four nucleotides at the 3′ terminus. In one embodiment, the sgRNA further comprises three PS bonds between the first four nucleotides at the 5′ terminus and three PS bonds between the last four nucleotides at the 3′ terminus.

In some embodiments, a sgRNA is provided comprising 2′-O-Me modified nucleotides at: the first three nucleotides in the 5′ terminus; LS1, LS6, LS7, LS8, LS11, and LS12 in the lower stem; B1-B6 in the bulge region; each of the nucleotides in the upper stem region; N16, N17, and N18 in the nexus region; each of the nucleotides in the hairpin 1 region; one nucleotide between hairpin 1 and hairpin 2; each of the nucleotides in the hairpin 2 region; and the last four nucleotides at the 3′ terminus. In one embodiment, the sgRNA further comprises three PS bonds between the first four nucleotides at the 5′ terminus and three PS bonds between the last four nucleotides at the 3′ terminus.

In some embodiments, a sgRNA is provided comprising 2′-F modified nucleotides at: LS9 and LS10 in the lower stem; 15-N18 in the nexus region; H2-9-HS-15 in the hairpin 2 region; and the second to last, third to last, and fourth to last nucleotide in the 3′ terminus region.

In some embodiments, a sgRNA is provided comprising 2′-F modified nucleotides at: each nucleotide in the lower stem; 15-N18 in the nexus region; H2-9-HS-15 in the hairpin 2 region; and the second to last, third to last, and fourth to last nucleotide in the 3′ terminus region.

In some embodiments, a single guide RNA (sgRNA) is provided comprising 2′-O-Me modified nucleotides at LS8, LS10, LS12, H1-2, H1-4, H1-6, H1-8, H1-10, H1-12, H2-1, H2-3, H2-5, H2-7, H2-9, H2-11, H2-13, H2-15, and the last and third to last nucleotides at the 3′ terminus; and 2′-F modifications at LS7, LS9, LS11; H1-1, H1-3, H1-5, H1-7, H1-9, H1-11, H1-13, H2-2, H2-4, H2-6, H2-8, H2-10, H2-12, H2-14, and the second to last and fourth to last nucleotide at the 3′ terminus.

Each of the following embodiments are encompassed:

-   -   Embodiment 01. A single guide RNA (sgRNA) comprising one or more         modifications in one or more of the following regions:         -   a. the 5′ terminus;         -   b. the lower stem region;         -   c. the bulge region;         -   d. the upper stem region;         -   e. the nexus region;         -   f. the hairpin 1 region;         -   g. the hairpin 2 region; and         -   h. the 3′ terminus.     -   Embodiment 02. The sgRNA of embodiment 1, wherein the         modification comprises a 2′-O-methyl (2′-O-Me) modified         nucleotide.     -   Embodiment 03. The sgRNA of embodiment 1, wherein the         modification comprises a 2′-fluoro (2′-F) modified nucleotide.     -   Embodiment 04. The sgRNA of embodiment 1, wherein the         modification comprises a phosphorothioate (PS) bond between         nucleotides.     -   Embodiment 05. The sgRNA of any one of embodiments 1-3, wherein         the first three or four nucleotides at the 5′ terminus, and the         last three or four nucleotides at the 3′ terminus are modified.     -   Embodiment 06. The sgRNA of any one of embodiments 1-5, wherein         the first four nucleotides at the 5′ terminus, and the last four         nucleotides at the 3′ terminus are linked with phosphorothioate         (PS) bonds.     -   Embodiment 07. The sgRNA of embodiment 5, wherein the         modification comprises 2′-O-Me.     -   Embodiment 08. The sgRNA of embodiment 5, wherein the         modification comprises 2′-F.     -   Embodiment 09. The sgRNA of any one of embodiments 1-7, wherein         the first four nucleotides at the 5′ terminus and the last four         nucleotides at the 3′terminus are linked with a PS bond, and         wherein the first three nucleotides at the 5′ terminus and the         last three nucleotides at the 3′ terminus comprise 2′-O-Me         modifications.     -   Embodiment 10. The sgRNA of any one of embodiments 1-8, wherein         the first four nucleotides at the 5′ terminus and the last four         nucleotides at the 3′ terminus are linked with a PS bond, and         wherein the first three nucleotides at the 5′ terminus and the         last three nucleotides at the 3′ terminus comprise 2′-F         modifications.     -   Embodiment 11. The sgRNA of any one of embodiments 1-10, wherein         LS1, LS6, LS7, LS8, LS11, and LS12 are modified with 2′-O-Me.     -   Embodiment 12. The sgRNA of any one of embodiments 1-11, wherein         each of the nucleotides in the bulge region are modified with         2′-O-Me.     -   Embodiment 13. The sgRNA of any one of embodiments 1-12, wherein         each of the nucleotides in the upper stem region are modified         with 2′-O-Me.     -   Embodiment 14. The sgRNA of any one of embodiments 1-13, wherein         N16, N17, and N18 in the nexus region are modified with 2′-O-Me.     -   Embodiment 15. The sgRNA of any one of embodiments 1-14, wherein         each of the nucleotides in the hairpin 1 region are modified         with 2′-O-Me.     -   Embodiment 16. The sgRNA of any one of embodiments 1-15, wherein         each of the nucleotides in the hairpin 2 region are modified         with 2′-O-Me.     -   Embodiment 17. A single guide RNA (sgRNA) comprising 2′-O-Me         modified nucleic acids at the following nucleotides:         -   a. the first three nucleotides at the 5′ terminus;         -   b. LS1, LS6, LS7, LS8, LS11, and LS12 in the lower stem             region;         -   c. B1 and B2 in the bulge region;         -   d. each nucleotide in the upper stem region;         -   e. N16, N17, and N18 in the nexus region;         -   f. each nucleotide in the hairpin 1 region;         -   g. each nucleotide in the hairpin 2 region; and         -   h. the last four nucleotides at the 3′ terminus.     -   Embodiment 18. The sgRNA of embodiment 17, wherein B3-B6 are         modified with 2′-O-Me.     -   Embodiment 19. The sgRNA of embodiment 17, further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus. Embodiment 20. The         sgRNA of any one of embodiments 1-10, wherein LS9 and LS10 are         modified with 2′-F.     -   Embodiment 21. The sgRNA of any one of embodiments 1-10 and 20,         wherein N15, N16, N17, and N18 are modified with 2′-F.     -   Embodiment 22. The sgRNA of any one of embodiments 1-10 and         20-21, wherein H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and         H2-15 are modified with 2′-F.     -   Embodiment 23. The sgRNA of any one of embodiments 1-10 and         21-22, wherein the second to last, third to last, and fourth to         last nucleotides at the 3′ terminus are modified with 2′-F.     -   Embodiment 24. A single guide RNA (sgRNA) comprising 2′-F         modified nucleotides at the following positions:         -   a. LS9 and LS10 in the lower stem region;         -   b. N15, N16, N17, and N18 in the nexus region; and         -   c. H2-9, H2-10, H2-11, H2-12, H2-13, H2-14, and H2-15 in the             hairpin 2 region.     -   Embodiment 25. The sgRNA of embodiment 24, further comprising         2′-F modified nucleotides at the second to last, third to last,         and fourth to last nucleotides at the 3′ terminus.     -   Embodiment 26. The sgRNA of any one of embodiments 24 or 25,         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ terminus and three PS bonds         linking the last four nucleotides at the 3′ terminus.     -   Embodiment 27. The sgRNA of any one of embodiments 24-26,         further comprising 2′-O-Me or 2′-F modified nucleotides at the         first three nucleotides at the 5′ terminus, and 2′-O-Me or 2′-F         modified nucleotides at the three of the last four nucleotides         at the 3′ terminus.     -   Embodiment 28. A single guide RNA (sgRNA) comprising         -   a. 2′-O-Me modified nucleotides at the first three             nucleotides at the 5′ terminus;         -   b. 2′-O-Me modified nucleotides at LS1 and LS6;         -   c. 2′-O-Me modified nucleotides at US1-US12;         -   d. 2′-O-Me modified nucleotides at H1-1-H1-12;         -   e. a 2′-O-Me modified nucleotide between Hairpin 1 and             Hairpin 2;         -   f. 2′-O-Me modified nucleotides at H2-1-H2-15; and         -   g. 2′-O-Me modified nucleotides at the last four nucleotides             at the 3′ terminus.     -   Embodiment 29. The sgRNA of embodiment 28 further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus.     -   Embodiment 30. A single guide RNA (sgRNA) comprising         -   a. 2′-O-Me modified nucleotides at the first three             nucleotides at the 5′ terminus;         -   b. 2′-F modified nucleotides at LS1-LS6;         -   c. 2′-O-Me modified nucleotides at US1-US12;         -   d. 2′-O-Me modified nucleotides at H1-1-H1-12;         -   e. a 2′-O-Me modified nucleotide between Hairpin 1 and             Hairpin 2;         -   f. 2′-O-Me modified nucleotides at H2-1-H2-15; and         -   g. 2′-O-Me modified nucleotides at the last four nucleotides             at the 3′ terminus.     -   Embodiment 31. The sgRNA of embodiment 30 further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus.     -   Embodiment 32. A single guide RNA (sgRNA) comprising         -   a. 2′-O-Me modified nucleotides at the first three             nucleotides at the 5′ terminus;         -   b. 2′-F modified nucleotides at LS2-LS5;         -   c. 2′-O-Me modified nucleotides at LS1 and LS6;         -   d. 2′-O-Me modified nucleotides at US1-US12;         -   e. 2′-O-Me modified nucleotides at H1-1-H1-12;         -   f. a 2′-O-Me modified nucleotide between Hairpin 1 and             Hairpin 2;         -   g. 2′-O-Me modified nucleotides at H2-1-H2-15; and         -   h. 2′-O-Me modified nucleotides at the last four nucleotides             at the 3′ terminus.     -   Embodiment 33. The sgRNA of embodiment 32 further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus.     -   Embodiment 34. A single guide RNA (sgRNA) comprising         -   a. 2′-O-Me modified nucleotides at the first three             nucleotides at the 5′ terminus;         -   b. 2′-O-Me modified nucleotides at US1-US12;         -   c. 2′-O-Me modified nucleotides at LS7, LS8, LS11, and LS12;         -   d. 2′-O-Me modified nucleotides at H1-1-H1-12;         -   e. a 2′-O-Me modified nucleotide between Hairpin 1 and             Hairpin 2;         -   f. 2′-O-Me modified nucleotides at H2-1-H2-15; and         -   g. 2′-O-Me modified nucleotides at the last four nucleotides             at the 3′ terminus.     -   Embodiment 35. The sgRNA of embodiment 34 further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus.     -   Embodiment 36. A single guide RNA (sgRNA) comprising         -   a. 2′-O-Me modified nucleotides at the first three             nucleotides at the 5′ terminus;         -   b. 2′-O-Me modified nucleotides at US1-US12;         -   c. 2′-O-Me modified nucleotides at LS7, LS8, LS11, and LS12;         -   d. 2′-F modified nucleotides at LS9 and LS10;         -   e. 2′-O-Me modified nucleotides at H1-1-H1-12;         -   f. a 2′-O-Me modified nucleotide between Hairpin 1 and             Hairpin 2;         -   g. 2′-O-Me modified nucleotides at H2-1-H2-15; and         -   h. 2′-O-Me modified nucleotides at the last four nucleotides             at the 3′ terminus.     -   Embodiment 37. The sgRNA of embodiment 36 further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus.     -   Embodiment 38. A single guide RNA (sgRNA) comprising         -   a. 2′-O-Me modified nucleotides at the first three             nucleotides at the 5′ terminus;         -   b. 2′-O-Me modified nucleotides at US1-US12;         -   c. 2′-O-Me modified nucleotides at LS8, LS10, and LS12;         -   d. 2′-O-F modified nucleotides at LS7, LS9, and LS11;         -   e. 2′-O-Me modified nucleotides at H1-1-H1-12;         -   f. a 2′-O-Me modified nucleotide between Hairpin 1 and             Hairpin 2;         -   g. 2′-O-Me modified nucleotides at H2-1-H2-15; and         -   h. 2′-O-Me modified nucleotides at the last four nucleotides             at the 3′ terminus.     -   Embodiment 39. The sgRNA of embodiment 32 further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus.     -   Embodiment 40. A single guide RNA (sgRNA) comprising         -   a. 2′-O-Me modified nucleotides at the first three             nucleotides at the 5′ terminus;         -   b. 2′-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11,             and LS12         -   c. 2′-O-Me modified nucleotides at US1-US12;         -   d. 2′-O-Me modified nucleotides at H1-1-H1-12;         -   e. a 2′-O-Me modified nucleotide between Hairpin 1 and             Hairpin 2;         -   f. 2′-O-Me modified nucleotides at H2-1-H2-15; and         -   g. 2′-O-Me modified nucleotides at the last four nucleotides             at the 3′ terminus.     -   Embodiment 41. The sgRNA of embodiment 40 further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus     -   Embodiment 42. A single guide RNA (sgRNA) comprising         -   a. 2′-O-Me modified nucleotides at the first three             nucleotides at the 5′ terminus;         -   b. 2′-O-Me modified nucleotides at LS1, LS6, LS7, LS8, LS11,             and LS12;         -   c. 2′-F modified nucleotides at LS9 and LS10;         -   d. 2′-O-Me modified nucleotides at US1-US12;         -   e. 2′-O-Me modified nucleotides at H1-1-H1-12;         -   f. a 2′-O-Me modified nucleotide between Hairpin 1 and             Hairpin 2;         -   g. 2′-O-Me modified nucleotides at H2-1-H2-15; and         -   h. 2′-O-Me modified nucleotides at the last four nucleotides             at the 3′ terminus.     -   Embodiment 43. The sgRNA of embodiment 43 further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus.     -   Embodiment 44. A single guide RNA (sgRNA) comprising         -   a. 2′-O-Me modified nucleotides at the first three             nucleotides at the 5′ terminus;         -   b. 2′-O-Me modified nucleotides at US1-US12;         -   c. 2′-O-Me modified nucleotides at H1-1-H1-12;         -   d. a 2′-O-Me modified nucleotide between Hairpin 1 and             Hairpin 2;         -   e. 2′-O-Me modified nucleotides at H2-1-H2-8;         -   f. 2′-F modified nucleotides at H2-9-H2-15;         -   g. 2′-F modified nucleotides at the second from last, third             from last, and fourth from last nucleotide at the 3′             terminus; and         -   h. a 2′-O-Me modified nucleotide at the last nucleotide at             the 3′ terminus.     -   Embodiment 45. The sgRNA of embodiment 44 further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus.     -   Embodiment 46. A single guide RNA (sgRNA) comprising         -   a. 2′-O-Me modified nucleotides at the first three             nucleotides at the 5′ terminus;         -   b. 2′-O-Me modified nucleotides at US1-US12;         -   c. 2′-O-Me modified nucleotides at H1-2, H1-4, H1-6, H1-8,             H1-10, and H1-12;         -   d. 2′-F modified nucleotides at H1-1, H1-3, H1-5, H1-7,             H1-9, and H1-11;         -   e. a 2′-F modified nucleotide between Hairpin 1 and Hairpin             2;         -   f. 2′-F modified nucleotides at H2-2, H2-4, H2-6, H2-8,             H2-10, H2-12; and H2-14;         -   g. 2′-O-Me modified nucleotides at H2-1, H2-3, H2-5, H2-7,             H2-9, H2-11; H2-13, and H2-15;         -   h. 2′-F modified nucleotides at the second from last, and             fourth from last nucleotide at the 3′ terminus; and         -   i. 2′-O-Me modified nucleotide at the third from last, and             last nucleotide at the 3′ terminus.     -   Embodiment 47. The sgRNA of embodiment 46 further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus.     -   Embodiment 48. A single guide RNA (sgRNA) comprising         -   a. 2′-O-Me modified nucleotides LS8, LS10, LS12, H1-2, H1-4,             H1-6, H1-8, H1-10, H1-12, H2-1, H2-3, H2-5, H2-7, H2-9,             H2-11, H2-13, and H2-15; and         -   b. 2′-F modified nucleotides at LS7, LS9, LS11; H1-1, H1-3,             H1-5, H1-7, H1-9, H1-11, H1-13, H2-2, H2-4, H2-6, H2-8,             H2-10, H2-12, and H2-14.     -   Embodiment 49. The sgRNA of embodiment 48, further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus.     -   Embodiment 50. The sgRNA of any one of embodiments 48-49,         further comprising         -   a. 2′-O-Me modified nucleotides at the last and third to             last nucleotide at the 3′ terminus; and         -   b. 2′-F modified nucleotides at the second to last and third             to last nucleotide at the 3′ terminus.     -   Embodiment 51. A sgRNA comprising the nucleic acids of any one         of SEQ ID Nos: 228-332.     -   Embodiment 52. A sgRNA comprising the nucleic acids of any one         of SEQ ID Nos: 235-240, 265-285, and 309-329.     -   Embodiment 53. A sgRNA comprising nucleic acids having at least         99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70%         identity to the nucleic acids of any one of SEQ ID Nos: 235-240,         265-285, and 309-329, wherein the modification pattern is         identical to the modification pattern shown in the reference         sequence identifier.     -   Embodiment 54. The sgRNA of any one of embodiments 51-53,         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ terminus and three PS bonds         linking the last four nucleotides at the 3′ terminus.

B. Compositions of dgRNAs

In some embodiments, the compositions and methods of the invention comprise gRNA comprising a crRNA and trRNA that direct a nuclease such as Cas9 to a target DNA sequence. In some embodiments, the gRNAs are associated, but on two separate RNA molecules (dual guide RNA or dgRNA).

Table 2 and FIG. 21C provides a description of domains of a crRNA as used herein. The 5′ terminus region may comprise a spacer region at or near the 5′ terminus of the crRNA and functions to direct a Cas9 to a target region in the DNA, e.g., as described herein. In Table 2, the “n” between regions represents a variable number of nucleotides, for example, from 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. In some embodiments, n equals 0. Any of the dgRNAs described herein may include an “n” between any domain.

Table 3 and FIG. 21C provide a description of domains of a trRNA as used herein. In Table 3, the “n” between regions represents a variable number of nucleotides, for example, from 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. In some embodiments, n equals 0. Any of the dgRNAs described herein may include an “n” between any domain.

1. Domains of dgRNAs

As described in Briner 2014, dgRNAs can be developed based on specific functional domains, referred to herein as “domains”, including the spacer responsible for targeting, the lower stem, the bulge, the upper stem, the nexus, and the hairpin domains. In dgRNAs, the crRNA comprises some components of the gRNA and the trRNA comprises some components of the gRNA.

Regions of crRNAs are provided in Table 2 and FIG. 21C. Regions of trRNAs are provided in Table 3 and FIG. 21C. FIG. 21C shows a schematic of an exemplary dgRNA.

TABLE 2 Regions of crRNA (linear view, 5′ to 3′) LS1-6 B1-2 US1-14 5′ terminus (n) lower stem n bulge n upper stem 3′ terminus

TABLE 3 Regions of trRNA (linear view, 5' to 3') H1-1 thru H2-1 thru US1-11 B1 -4 LS1-6 N1-18 H1-12 H2-15 5′ terminus upper n bulge n lower n nexus n hairpin 1 n hairpin 2 3′ (n) stem stem terminus

a) 5′ Terminus Region

In some embodiments, the dgRNA comprises nucleotides at the 5′ terminus of the crRNA and trRNA as shown in Tables 2-3 and FIG. 21C.

In some embodiments, the 5′ terminus of the crRNA comprises a spacer or guide region that functions to direct a Cas protein to a target nucleotide sequence. In some embodiments, the 5′ terminus does not comprise a spacer or guide region. In some embodiments, the 5′ terminus comprises a spacer and additional nucleotides that do not function to direct a Cas protein to a target nucleotide region.

In some embodiments, the guide region comprises the first 1-10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides at the 5′ end of the crRNA. In some embodiments, the guide region comprises 20 nucleotides. In some embodiments, the guide region may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more nucleotides. In some embodiments, the guide region may comprise 17 nucleotides. In some embodiments, the guide region may comprise 18 nucleotides. In some embodiments, the guide region may comprise 19 nucleotides.

In some embodiments, the selection of the guide region is determined based on target sequences within the gene of interest for editing. For example, in some embodiments, the crRNA comprises a guide region that is complementary to target sequences of a gene of interest.

In some embodiments, the target sequence in the gene of interest may be complementary to the guide region of the crRNA. In some embodiments, the degree of complementarity or identity between a guide region of a crRNA and its corresponding target sequence in the gene of interest may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide region of a crRNA and the target region of a gene of interest may be 100% complementary or identical. In other embodiments, the guide region of a crRNA and the target region of a gene of interest may contain at least one mismatch. For example, the guide region of a crRNA and the target sequence of a gene of interest may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches, where the total length of the target sequence is at least about 17, 18, 19, 20 or more base pairs. In some embodiments, the guide region of a crRNA and the target region of a gene of interest may contain 1-6 mismatches where the guide sequence comprises at least about 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide region of a crRNA and the target region of a gene of interest may contain 1, 2, 3, 4, 5, or 6 mismatches where the guide sequence comprises about 20 nucleotides.

In some embodiments, the trRNA comprises a 5′ terminus. In some embodiments, the trRNA comprises a 5′ terminus which forms, in part, the upper stem of a dgRNA. The 5′ terminus of the trRNA is not complementary to a region of the target gene.

b) Lower stem

In some embodiments, the dgRNA comprises a lower stem (LS) region. The lower stem region comprises a crRNA lower stem region and a trRNA lower stem region that associate as depicted in FIG. 21C. In some embodiments, the lower stem region of the crRNA is at least partially complementary to the lower stem region of the trRNA. In some embodiments, the lower stem region of the crRNA is fully complementary to the lower stem region of the trRNA.

In some embodiments, the lower stem region of the crRNA and trRNA each comprise 6 nucleotides. In some embodiments, the lower stem region of the crRNA and trRNA each comprise fewer nucleotides than shown in Tables 2 and 3 and FIG. 21C. In some embodiments, the lower stem region comprises more nucleotides than shown in Tables 2 and 3 and FIG. 21C. When the lower stem region comprises fewer or more nucleotides than shown in the schematic of Tables 2 and 3 and FIG. 21C, the modification patterns, as will be apparent to the skilled artisan, are maintained. In some embodiments, the number of nucleotides in the lower stem of the crRNA differs from the number of nucleotides in the lower stem of the trRNA.

c) Bulge

In some embodiments, the dgRNA comprises a bulge (B) region. In some embodiments, the crRNA comprises one bulge region and the trRNA comprises one bulge region. In some embodiments, each bulge region comprises 1-4 nucleotides. In some embodiments, the bulge region of the crRNA comprises two nucleotides, and the bulge region of the trRNA comprises four nucleotides.

In some embodiments, the crRNA bulge region is located between the lower stem region and the upper stem region of the crRNA. In some embodiments, the bulge region of the crRNA comprises two nucleotides. In some embodiments, the bulge region of the crRNA comprises nucleotides B1 and B2 as shown Table 2 and FIG. 21C.

In some embodiments, the trRNA bulge region is located between the upper stem region and the lower stem region of the trRNA. In some embodiments, the bulge region of the trRNA comprises four nucleotides. In some embodiments, the bulge region of the trRNA comprises nucleotides B1 through B4 as shown Table 3 and FIG. 21C.

In some embodiments, the presence of a bulge results in a directional kink between the upper and lower stems modules in a dgRNA. The crRNA bulge and trRNA bulge may be partially complementary. The crRNA bulge and trRNA bulge may have no complementary.

In some embodiments, the bulge regions of the crRNA and trRNA comprise more nucleotides than shown in Tables 2 and 3 and FIG. 21C. When the bulge region comprises fewer or more nucleotides than shown in the schematic of Tables 2 and 3 and FIG. 21C, the modification patterns, as will be apparent to the skilled artisan, are maintained. In some embodiments, the number of nucleotides in the bulge of the crRNA differs from the number of nucleotides in the bulge of the trRNA.

-   -   d) Upper stem

In some embodiments, the dgRNA comprises an upper stem (US) region. The upper stem region comprises a crRNA upper stem region and a trRNA upper stem region that associate as depicted in FIG. 21C. In some embodiments, the upper stem region of the crRNA is at least partially complementary to the upper stem region of the trRNA. In some embodiments, the upper stem region of the crRNA is fully complementary to the upper stem region of the trRNA.

In some embodiments, the upper stem region of the crRNA comprises fourteen nucleotides. In some embodiments, the upper stem region of the trRNA comprises eleven nucleotides. In some embodiments, the upper stem regions of the crRNA and trRNA each comprise fewer nucleotides than shown in Tables 2 and 3 and FIG. 21C. In some embodiments, the upper stem regions of the crRNA and trRNA comprise more nucleotides than shown in Tables 2 and 3 and FIG. 21C. When the upper stem region comprises fewer or more nucleotides than shown in the schematic of Tables 2 and 3 and FIG. 21C, the modification patterns, as will be apparent to the skilled artisan, are maintained.

In some embodiments, the upper stem of the crRNA comprises nucleotides US1 through US14 as shown in Table 2 and FIG. 21C.

In some embodiments, the upper stem of the trRNA comprises nucleotides US1 through US11 as shown in Table 3 and FIG. 21C.

e) Nexus

In some embodiments, the dgRNA comprises a trRNA comprising a nexus region. In some embodiments, the nexus is between the lower stem region and the hairpin 1 region of the trRNA. In some embodiments, the nexus is located immediately downstream of the lower stem of the trRNA. In some embodiments, the nexus comprises eighteen nucleotides. In some embodiments, the nexus region of the trRNA comprises nucleotides N1-N18 as shown in Table 3 and FIG. 21C. In some embodiments, the nexus comprises fewer nucleotides than shown in Table 3 and FIG. 21C. In some embodiments, the nexus region of the trRNA comprises more nucleotides than shown in Table 3 and FIG. 21C. When the nexus region comprises fewer or more nucleotides than shown in Table 3 and FIG. 21C, the modification patterns, as will be apparent to the skilled artisan, are maintained.

In some embodiments, the nexus region has nucleotides that are complementary in nucleic acid sequence when read in opposite directions. In some embodiments, the complementarity in nucleic acid sequence leads to a secondary structure of a stem and/or stem loop in the sgRNA (e.g., certain nucleotides in the nexus region may base pair with one another). In some embodiments, the nexus regions may not be perfectly complimentary to each other when read in opposite directions.

f) Hairpin

In some embodiments, the hairpin region of the trRNA is downstream of the nexus region. In some embodiments, the region of nucleotides immediately downstream of the nexus region is termed “hairpin 1.” In some embodiments, the region of nucleotides immediately downstream of the hairpin 1 region is termed “hairpin 2.” In some embodiments, the hairpin region comprises hairpin 1 and hairpin 2. In some instances, hairpin 1 and hairpin 2 are separated by one or more nucleotide “n.” In some embodiments, n=1. In some embodiments, the trRNA comprises only hairpin 1 or hairpin 2.

Replacement of the hairpin 1 region of a trRNA with 2 nucleotides has been shown to allow editing activity of a Cas RNP (see US20150376586, FIG. 16 ). In some embodiments, the trRNA comprises replacement of hairpin 1 with nucleotides “n”, wherein “n” is an integer between 1 and 50, 40, 30, 20, 15, 10, 5, 4, 3, and 2. In some embodiments, the hairpin 1 region of a trRNA is replaced by 2 nucleotides.

In some embodiments, hairpin 1 of the trRNA comprises twelve nucleotides immediately downstream of the nexus region. In some embodiments, the hairpin 1 region of the trRNA comprises nucleotides H1-1 through H1-12 as shown in Table 3 and FIG. 21C.

In some embodiments, non-hairpin nucleotides are present between the hairpin 1 and the hairpin 2 regions of the trRNA. In some embodiments, one to two non-hairpin nucleotides reside between hairpin 1 and hairpin 2.

In some embodiments, hairpin 2 of the trRNA comprises fifteen nucleotides after (3′ to) hairpin 1. In some embodiments, the hairpin 2 region of the trRNA comprises nucleotides H2-1 through H2-15 as shown in Table 3 and FIG. 21C. In some embodiments, the hairpin 2 region of the trRNA comprises nucleotides H2-1 through H2-15 as shown in Table 3, and the “n” between hairpin 1 and hairpin 2 is 1 or 2.

In some embodiments, a hairpin region of the trRNA comprises more nucleotides than shown in Table 3 and FIG. 21C. When a hairpin region comprises fewer or more nucleotides than shown in Table 3 and FIG. 21C, the modification patterns, as will be apparent to the skilled artisan, are maintained.

In some embodiments, a hairpin region has nucleotides that are complementary in nucleic acid sequence when read in opposite directions. In some embodiments, the hairpin regions may not be perfectly complimentary to each other when read in opposite directions (e.g., the top or loop of the hairpin comprises unpaired nucleotides).

In some embodiments, the trRNA comprises replacement of hairpin 1 with nucleotides “n”, wherein “n” is an integer between 1 and 50, 40, 30, 20, 15, 10, 5, 4, 3, and 2. In some embodiments, the hairpin 1 region of a trRNA is replaced by 2 nucleotides.

g) 3′ terminus

In some embodiments, the dgRNA comprises a trRNA comprising a 3′ terminus region comprising additional nucleotides after (3′ to) the hairpin region(s). In some embodiments, the 3′ terminus region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 or more nucleotides that are not associated with the secondary structure of a hairpin. In some embodiments, the 3′ terminus region comprises 1, 2, 3, or 4 nucleotides that are not associated with the secondary structure of a hairpin. In some embodiments, the 3′ terminus region comprises 4 nucleotides that are not associated with the secondary structure of a hairpin. In some embodiments, the 3′ terminus region comprises 1, 2, or 3 nucleotides that are not associated with the secondary structure of a hairpin.

2. Modifications of dgRNAs

In some embodiments, a dgRNA comprises a modified crRNA and an unmodified trRNA. In some embodiments, a dgRNA comprises an unmodified crRNA and a modified trRNA. In some embodiments, both the crRNA and trRNA of a dgRNA comprise modifications.

In some embodiments, the gRNAs described herein are in two separate RNA molecules (dual guide or dgRNA). See, Tables 2, 3, and FIG. 21C.

In some embodiments, the invention comprises a dgRNA comprising or consisting of a) any one of the crRNA sequences of SEQ ID Nos: 1-187; and b) any one of the trRNA sequences described in SEQ ID Nos: 188-227.

In some embodiments, a dgRNA comprising any one of the modified crRNA sequences of 1-187 is provided.

In some embodiments, a dgRNA comprising any one of the modified trRNA sequences of 188-227 is provided.

In some embodiments, a dgRNA comprising any one of the modified crRNA sequences of SEQ ID Nos: 19-31, 53-73, and 104-130 is provided. In some embodiments, the invention comprises a dgRNA comprising any one of the modified sequences of SEQ ID Nos: 19-31, 53-73, and 104-130, wherein the crRNA further comprises a 5′ spacer sequence that is at least partially complementary to a target sequence, and directs a Cas9 to its target for cleavage.

In some embodiments, the invention comprises a crRNA comprising any one of the sequences described in SEQ ID Nos: 1-187. In some embodiments, the invention comprises a crRNA comprising or consisting of any one of the sequences described in SEQ ID Nos: 19-31, 53-73, and 104-130. In some embodiments, the invention comprises a crRNA comprising any one of the sequences described in SEQ ID Nos: 19-31, 53-73, and 104-130 and a spacer region.

In some embodiments, the invention comprises a trRNA comprising or consisting of any one of the sequences described in SEQ ID Nos:188-277.

In some embodiments, the invention comprises a crRNA comprising nucleotides having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to the nucleotides of any one of SEQ ID Nos: 1-187, wherein the modification pattern is identical to the modification pattern shown in the reference sequence identifier. That is, the nucleotides A, U, C, and G may differ by 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% compared to what is shown in in the sequences, but the modification remains unchanged.

In some embodiments, the invention comprises a trRNA comprising nucleotides having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to the nucleotides of any one of SEQ ID Nos: 188-277, wherein the modification pattern is identical to the modification pattern shown in the reference sequence identifier. That is, the nucleotides A, U, C, and G may differ by 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% compared to what is shown in the sequences, but the modification on each nucleotide remains unchanged.

3. crRNAs, trRNAs, and dgRNAs with modifications

In some embodiments, the crRNA comprises one or more modified nucleotides within one or more of the 5′ terminus, lower stem, bulge, upper stem, and 3′ terminus.

In some embodiments, the modification comprises 2′-O-Me.

In some embodiments, the modification comprises 2′-F.

In some embodiments, the modification comprises a phosphorothioate (PS) bond linking one or more nucleotides. In some embodiments, the modification is three PS bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

In some embodiments, the modification comprises an inverted abasic nucleotide.

In some embodiments, a crRNA is provided comprising 2′-O-Me modified nucleotides at each nucleotide in the upper stem. In some embodiments, US-1 through US-14 of the crRNA are each modified with 2′-O-Me. In some embodiments, LS1 and LS6 of the crRNA are modified with 2′-O-Me. In some embodiments, LS5 of the crRNA is modified with 2′-O-Me.

In some embodiments, a crRNA comprising 2′-O-Me modified nucleotides at each of the nucleotides in the upper stem, and LS1 and LS6 in the lower stem is provided. In some embodiments, the crRNA further comprises one or more 2′-O-Me or 2′-O-moe modified nucleotides in the 5′ and/or 3′ terminus region, e.g. in a 5′ and/or 3′ end modification.

In some embodiments, a crRNA comprising 2′-O-Me modified nucleotides at each of the nucleotides in the upper stem, LS1, LS5, and LS6 in the lower stem is provided. In some embodiments, the crRNA further comprises one or more 2′-O-Me or 2′-O-moe modified nucleotides in the 5′ and/or 3′ terminus region, e.g. in a 5′ and/or 3′ end modification.

In some embodiments, the invention comprises a crRNA comprising 2′-F modified nucleotides at LS1, LS2, and LS6 in the lower stem. In some embodiments, the crRNA further comprises 2′-F modified nucleotides at each of B1 and B2 in the bulge region. In some embodiments, the invention comprises a crRNA comprising 2′-F modified nucleotides at LS1, LS2, and LS6 in the lower stem, and at each of B1 and B2 in the bulge region. In some embodiments, the crRNA further comprises one or more 2′-O-Me or 2′-O-moe modified nucleotides in the 5′ and/or 3′ terminus region, e.g. in a 5′ and/or 3′ end modification.

In some embodiments, the crRNA comprises 2′-O-Me modified nucleotides at nucleotides LS1 and LS6 in the lower stem region; each of the nucleic acids in the bulge region; and each of the nucleic acids in the upper stem region. In some embodiments, the LS5 nucleotide of the crRNA is also modified with 2′-O-Me. In some embodiments, LS2, LS3, and LS4 of the crRNA are not modified. In some embodiments, the crRNA further comprises one or more 2′-O-Me or 2′-O-moe modified nucleotides in the 5′ and/or 3′ terminus region, e.g. in a 5′ and/or 3′ end modification.

In some embodiments, the crRNA comprises 2′-fluoro (2′-F) modified nucleotides at LS1, LS2, and LS6 in the lower stem region, and each of the nucleotides in the bulge region. In some embodiments, the crRNA comprises 2′-fluoro (2′-F) modified nucleotides at LS1, LS2, and LS6 in the lower stem region, and at B2 and B2 in the bulge region. In some embodiments, the crRNA comprises 2′-fluoro (2′-F) modified nucleotides at LS1-LS6 in the lower stem region, and each of the nucleotides in the bulge region. In some embodiments, the crRNA further comprises one or more 2′-O-Me or 2′-O-moe modified nucleotides in the 5′ and/or 3′ terminus region, e.g. in a 5′ and/or 3′ end modification.

In some embodiments, the invention comprises a trRNA comprising one or more modified nucleotides within one or more of the following regions: the 5′ terminus, the upper stem region; the bulge region; the lower stem region; the nexus region; the hairpin 1 region; the intervening region between the hairpin 1 and hairpin 2 regions; the hairpin 2 region; and the 3′ terminus region.

In some embodiments, the modification comprises 2′-O-Me.

In some embodiments, the modification comprises 2′-F.

In some embodiments, the modification comprises a phosphorothioate (PS) bond linking one or more nucleotides. In some embodiments, the modification is three PS bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

In some embodiments, the modification comprises an inverted abasic nucleotide.

In some embodiments, the trRNA comprises 2′-O-Me modified nucleotides at each nucleic acid in the upper stem; B1 and B2 in the bulge region; LS1 and LS2 in the lower stem region; N3, N4, N5, N15, N16, N17, and N18 in the nexus region; each nucleotide in the hairpin 1 region; one nucleotide between the hairpin 1 and hairpin 2 region; and each nucleotide in the hairpin 2 region. In some embodiments, the trRNA further comprises one or more 2′-O-Me or 2′-O-moe modified nucleotides in the 5′ and/or 3′ terminus region, e.g. in a 5′ and/or 3′ end modification.

In some embodiments, the trRNA comprises 2′-O-Me modified nucleotides at each nucleic acid in the upper stem; each nucleotide in the bulge region; LS1, LS2, LS5, and LS6 in the lower stem region; N3-N5, N10-N18 in the nexus region; each nucleotide in the hairpin 1 region; one nucleotide between the hairpin 1 and hairpin 2 region; and each nucleotide in the hairpin 2 region. In some embodiments, the crRNA further comprises one or more 2′-O-Me or 2′-O-moe modified nucleotides in the 5′ and/or 3′ terminus region, e.g. in a 5′ and/or 3′ end modification.

In some embodiments, the trRNA comprises 2′-F modified nucleotides at N15 through N18 in the nexus region. In some embodiments, the trRNA further comprises one or more 2′-F modified nucleotides in the 5′ and/or 3′ terminus region, e.g. in a 5′ and/or 3′ end modification.

In some embodiments, the trRNA comprises 2′-F modified nucleotides at LS4 and LS5 in the lower stem region, and N13-N18 in the nexus region. In some embodiments, the trRNA further comprises one or more 2′-F modified nucleotides in the 5′ and/or 3′ terminus region, e.g. in a 5′ and/or 3′ end modification.

In some embodiments, the trRNA comprises 2′-F modified nucleotides at LS1, LS3, and LS5 in the lower stem, and 2′-O-Me modified nucleotides at LS2, LS4, and LS6 in the lower stem.

Disclosed herein, in some embodiments, is a crispr RNA (crRNA) comprising one or more modifications within one or more of the following regions: the first five nucleotides at the 5′ terminus; the lower stem region; the bulge region; the upper stem region; and the last five nucleotides at the 3′ terminus. In some embodiments, the modification comprises a 2′-O-methyl (2′-O-Me) modified nucleotide. In some embodiments, the modification comprises a 2′-fluoro (2′-F) modified nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the first three nucleotides at the 5′ terminus, and the last three nucleotides at the 3′ terminus are modified. In some embodiments, the first four nucleotides at the 5′ terminus, and the last four nucleotides at the 3′ terminus are linked with phosphorothioate (PS) bonds. In some embodiments, the modification comprises 2′-O-Me. In some embodiments, the modification comprises 2′-F. In some embodiments, the first four nucleotides at the 5′ terminus and the last four nucleotides at the 3′ terminus are linked with a PS bond, and wherein the first three nucleotides at the 5′ terminus and the last three nucleotides at the 3′ terminus comprise 2′-O-Me modifications. In some embodiments, the first four nucleotides at the 5′ terminus and the last four nucleotides at the 3′ terminus are linked with a PS bond, and wherein the first three nucleotides at the 5′ terminus and the last three nucleotides at the 3′ terminus comprise 2′-F modifications. In some embodiments, LS1 and LS6 are modified with 2′-O-Me. In some embodiments, each of the nucleotides in the upper stem region are modified with 2′-O-Me.

In some embodiments, the invention comprises a crispr RNA (crRNA) comprising 2′-O-Me modified nucleic acids at the following nucleotides: LS1 and LS6 in the lower stem region; and each nucleotide in the upper stem region. In some embodiments, the crRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus. In some embodiments, the crRNA further comprises 2′-O-Me or 2′-F modified nucleic acids at the first three nucleotides at the 5′ terminus, and 2′-O-Me or 2′-F modified nucleic acids at the last three nucleotides at the 3′ terminus. In some embodiments, LS1, LS2, and LS6 are modified with 2′-F. In some embodiments, each nucleotide in the bulge region is modified with 2′-F.

Disclosed herein, in some embodiments, is a crispr RNA (crRNA) comprising 2′-F modified nucleic acids at the following nucleotides: LS1, LS2, and LS6 in the lower stem region; and each nucleotide in the bulge region. In some embodiments, the crRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus. In some embodiments, the crRNA further comprises 2′-O-Me or 2′-F modified nucleic acids at the first three nucleotides at the 5′ terminus, and 2′-O-Me or 2′-F modified nucleic acids at the last three nucleotides at the 3′ terminus.

In some embodiments, a crRNA comprising the nucleic acids of any one of SEQ ID Nos: 1-187 is provided. In some embodiments, a crRNA comprising the nucleic acids of any one of SEQ ID Nos: 19-31, 53-73, 104-130, and 161-187 is provided. In some embodiments, a crRNA comprising nucleic acids having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to the nucleic acids of any one of SEQ ID Nos: 19-31, 53-73, 104-130, and 161-187, wherein the modification pattern is identical to the modification pattern shown in the reference sequence identifier, is provided. In some embodiments, the crRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

Also encompassed is a tracr RNA (trRNA) comprising one or more modifications within one or more of the following regions: the first five nucleotides at the 5′ terminus; the upper stem region; the bulge region; the lower stem region; the nexus region; the hairpin 1 region; the hairpin 2 region; and the last five nucleotides at the 3′ terminus. In some embodiments, the modification comprises a 2′-O-methyl (2′-O-Me) modified nucleotide. In some embodiments, the modification comprises a 2′-fluoro (2′-F) modified nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the first four nucleotides at the 5′ terminus, and the last four nucleotides at the 3′ terminus are linked with phosphorothioate (PS) bonds. In some embodiments, the first three nucleotides at the 5′ terminus, and the last three nucleotides at the 3′ terminus are modified. In some embodiments, the modification comprises 2′-O-Me. In some embodiments, the modification comprises 2′-F. In some embodiments, the first four nucleotides at the 5′ terminus and the last four nucleotides at the 3′ terminus are linked with a PS bond, and wherein the first three nucleotides at the 5′ terminus and the last three nucleotides at the 3′ terminus comprise 2′-O-Me modifications. In some embodiments, the first four nucleotides at the 5′ terminus and the last four nucleotides at the 3′ terminus are linked with a PS bond, and wherein the first three nucleotides at the 5′ terminus and the last three nucleotides at the 3′ terminus comprise 2′-F modifications. In some embodiments, each nucleotide in the upper stem region is modified with 2′-O-Me. In some embodiments, B1 and B2 within the bulge region are modified with 2′-O-Me. In some embodiments, N3, N4, N5, N15, N16, N17, and N18 in the nexus region are modified with 2′-O-Me. In some embodiments, each nucleotide in the hairpin 1 region is modified with 2′-O-Me. In some embodiments, each nucleotide in the hairpin 2 region is modified with 2′-O-Me.

In some embodiments, the invention comprises a tracr RNA (trRNA) comprising 2′-O-Me modified nucleic acids at the following nucleotides: each nucleotide in the upper stem; B1 and B2 within the bulge region; N3, N4, N5, N15, N16, N17, and N18 in the nexus region; each nucleotide in the hairpin 1 region; and each nucleotide in the hairpin 2 region. In some embodiments, the trRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus. In some embodiments, the trRNA further comprises 2′-O-Me or 2′-F modified nucleotides at the first three nucleotides at the 5′ terminus, and 2′-O-Me or 2′-F modified nucleic acids at the last three nucleotides at the 3′ terminus. In some embodiments, N15, N16, N17, and N18 are modified with 2′-F. In some embodiments, LS1, LS3, and LS5 are modified with 2′-F, and LS2, LS4, and LS6 are modified with 2′-O-Me. In some embodiments, the trRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus. In some embodiments, the trRNA further comprises 2′-O-Me or 2′-F modified nucleic acids at the first three nucleotides at the 5′ terminus, and 2′-O-Me or 2′-F modified nucleic acids at the last three nucleotides at the 3′ terminus.

In some embodiments, a trRNA comprising the nucleic acids of any one of SEQ ID Nos: 188-227 is provided. In some embodiments, a trRNA comprising nucleic acids having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70% identity to the nucleic acids of any one of SEQ ID Nos: 188-227, wherein the modification pattern is identical to the modification pattern shown in the reference sequence identifier, is provided. In some embodiments, the trRNA further comprises three phosphorothioate (PS) bonds linking the first four nucleotides at the 5′ terminus and three PS bonds linking the last four nucleotides at the 3′ terminus.

In some instances, a dual guide comprising a crRNA and a trRNA is provided, wherein the crRNA comprises the nucleic acids of any one of SEQ ID Nos: 1-187, and wherein the trRNA comprises the nucleic acids of any one of SEQ ID Nos: 188-227.

A dual guide comprising a crRNA disclosed herein and a trRNA disclosed herein is encompassed, as is a dual guide comprising a crRNA disclosed herein and an unmodified trRNA. In some embodiments, a dual guide comprising an unmodified crRNA and a modified trRNA disclosed herein is provided.

In some embodiments, and of the following are encompassed:

-   -   Embodiment 55. A crispr RNA (crRNA) comprising one or more         modifications within one or more of the following regions:         -   a. the first five nucleotides at the 5′ terminus;         -   b. the lower stem region;         -   c. the bulge region;         -   d. the upper stem region; and         -   e. the last five nucleotides at the 3′ terminus.     -   Embodiment 56. The crRNA of embodiment 55, wherein the         modification comprises a 2′-O-methyl (2′-O-Me) modified         nucleotide.     -   Embodiment 57. The crRNA of embodiment 55, wherein the         modification comprises a 2′-fluoro (2′-F) modified nucleotide.     -   Embodiment 58. The crRNA of embodiment 55, wherein the         modification comprises a phosphorothioate (PS) bond between         nucleotides.     -   Embodiment 59. The crRNA of any one of embodiments 55-58,         wherein the first three nucleotides at the 5′ terminus and the         last three nucleotides at the 3′ terminus are modified.     -   Embodiment 60. The crRNA of any one of embodiments 55-58,         wherein the first four nucleotides at the 5′ terminus, and the         last four nucleotides at the 3′ terminus are linked with         phosphorothioate (PS) bonds.     -   Embodiment 61. The crRNA of embodiment 59, wherein the         modification comprises 2′-O-Me.     -   Embodiment 62. The crRNA of embodiment 59, wherein the         modification comprises 2′-F.     -   Embodiment 63. The crRNA of any one of embodiments 55-62,         wherein the first four nucleotides at the 5′ terminus and the         last four nucleotides at the 3′terminus are linked with a PS         bond, and wherein the first three nucleotides at the 5′ terminus         and the last three nucleotides at the 3′ terminus comprise         2′-O-Me modifications.     -   Embodiment 64. The crRNA of any one of embodiments 55-62,         wherein the first four nucleotides at the 5′ terminus and the         last four nucleotides at the 3′terminus are linked with a PS         bond, and wherein the first three nucleotides at the 5′ terminus         and the last three nucleotides at the 3′ terminus comprise 2′-F         modifications.     -   Embodiment 65. The crRNA of any one of embodiments 55-60,         wherein LS1 and LS6 are modified with 2′-O-Me.     -   Embodiment 66. The crRNA of any one of embodiments 55-60 and 65,         wherein each of the nucleotides in the upper stem region are         modified with 2′-O-Me.     -   Embodiment 67. A crispr RNA (crRNA) comprising 2′-O-Me modified         nucleotides at:         -   a. LS1 and LS6 in the lower stem region; and         -   b. each nucleotide in the upper stem region.     -   Embodiment 68. The crRNA of embodiment 67, further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus.     -   Embodiment 69. The crRNA of embodiment 67 or 68, further         comprising 2′-O-Me or 2′-F modified nucleotides at the first         three nucleotides at the 5′ terminus, and 2′-O-Me or 2′-F         modified nucleotides at the last three nucleotides at the 3′         terminus.     -   Embodiment 70. The crRNA of any of embodiments 55-60, wherein         LS1, LS2, and LS6 are modified with 2′-F.     -   Embodiment 71. The crRNA of any of embodiments 55-60 and 70,         wherein each nucleotide in the bulge region is modified with         2′-F.     -   Embodiment 72. A crispr RNA (crRNA) comprising 2′-F modified         nucleotides at:         -   a. LS1, LS2, and LS6 in the lower stem region; and         -   b. each nucleotide in the bulge region.     -   Embodiment 73. The crRNA of any one of embodiments 70-72,         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ terminus and three PS bonds         linking the last four nucleotides at the 3′ terminus.     -   Embodiment 74. The crRNA of embodiment 72 or 73, further         comprising 2′-O-Me or 2′-F modified nucleotides at the first         three nucleotides at the 5′ terminus, and 2′-O-Me or 2′-F         modified nucleotides at the last three nucleotides at the 3′         terminus.     -   Embodiment 75. A crRNA comprising the nucleic acids of any one         of SEQ ID Nos: 1-187.     -   Embodiment 76. A crRNA comprising the nucleic acids of any one         of SEQ ID Nos: 19-31, 53-73, 104-130, and 161-187.     -   Embodiment 77. A crRNA comprising nucleic acids having at least         99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70%         identity to the nucleic acids of any one of SEQ ID Nos: 19-31,         53-73, 104-130, and 161-187, wherein the modification pattern is         identical to the modification pattern shown in the reference         sequence identifier.     -   Embodiment 78. The crRNA of any one of embodiments 75-77,         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ terminus and three PS bonds         linking the last four nucleotides at the 3′ terminus.     -   Embodiment 79. A tracr RNA (trRNA) comprising one or more         modifications within one or more of the following regions:         -   a. the first five nucleotides at the 5′ terminus;         -   b. the upper stem region;         -   c. the bulge region;         -   d. the lower stem region;         -   e. the nexus region;         -   f. the hairpin 1 region;         -   g. the hairpin 2 region; and         -   h. the last five nucleotides at the 3′ terminus.     -   Embodiment 80. The trRNA of embodiment 79, wherein the         modification comprises a 2′-O-methyl (2′-O-Me) modified         nucleotide.     -   Embodiment 81. The trRNA of embodiment 79, wherein the         modification comprises a 2′-fluoro (2′-F) modified nucleotide.     -   Embodiment 82. The trRNA of embodiment 79, wherein the         modification comprises a phosphorothioate (PS) bond between         nucleotides.     -   Embodiment 83. The trRNA of any one of embodiments 79-82,         wherein the first four nucleotides at the 5′ terminus, and the         last four nucleotides at the 3′ terminus are linked with         phosphorothioate (PS) bonds.     -   Embodiment 84. The trRNA of any one of embodiments 79-82,         wherein the first three nucleotides at the 5′ terminus, and the         last three nucleotides at the 3′ terminus are modified.     -   Embodiment 85. The trRNA of embodiment 84, wherein the         modification comprises 2′-O-Me.     -   Embodiment 86. The trRNA of embodiment 84, wherein the         modification comprises 2′-F.     -   Embodiment 87. The trRNA of any one of embodiments 79-86,         wherein the first four nucleotides at the 5′ terminus and the         last four nucleotides at the 3′terminus are linked with a PS         bond, and wherein the first three nucleotides at the 5′ terminus         and the last three nucleotides at the 3′ terminus comprise         2′-O-Me modifications.     -   Embodiment 88. The trRNA of any one of embodiments 79-86,         wherein the first four nucleotides at the 5′ terminus and the         last four nucleotides at the 3′terminus are linked with a PS         bond, and wherein the first three nucleotides at the 5′ terminus         and the last three nucleotides at the 3′ terminus comprise 2′-F         modifications.     -   Embodiment 89. The trRNA of any one of embodiments 79-84,         wherein each nucleotide in the upper stem region is modified         with 2′-O-Me.     -   Embodiment 90. The trRNA of any one of embodiments 79-84 and 89,         wherein B1 and B2 within the bulge region are modified with         2′-O-Me.     -   Embodiment 91. The trRNA of any one of embodiments 79-84 and         89-90, wherein N3, N4, N5, N15, N16, N17, and N18 in the nexus         region are modified with 2′-O-Me.     -   Embodiment 92. The trRNA of any one of embodiments 79-84 and         89-91, wherein each nucleotide in the hairpin 1 region is         modified with 2′-O-Me.     -   Embodiment 93. The trRNA of any one of embodiments 79-84 and         89-92, wherein each nucleotide in the hairpin 2 region is         modified with 2′-O-Me.     -   Embodiment 94. A tracr RNA (trRNA) comprising 2′-O-Me modified         nucleotides at:         -   a. each nucleotide in the upper stem;         -   b. B1 and B2 within the bulge region;         -   c. N3, N4, N5, N15, N16, N17, and N18 in the nexus region;         -   d. each nucleotide in the hairpin 1 region; and         -   e. each nucleotide in the hairpin 2 region.     -   Embodiment 95. The trRNA of embodiment 94, further comprising         three phosphorothioate (PS) bonds linking the first four         nucleotides at the 5′ terminus and three PS bonds linking the         last four nucleotides at the 3′ terminus.     -   Embodiment 96. The crRNA of embodiment 94 or 95, further         comprising 2′-O-Me or 2′-F modified nucleotides at the first         three nucleotides at the 5′ terminus, and 2′-O-Me or 2′-F         modified nucleic acids at the last three nucleotides at the 3′         terminus.     -   Embodiment 97. The trRNA of any of embodiments 79-84, wherein         N15, N16, N17, and N18 are modified with 2′-F.     -   Embodiment 98. The trRNA of any of embodiments 79-84 and 97,         wherein LS1, LS3, and LS5 are modified with 2′-F, and LS2, LS4,         and LS6 are modified with 2′-O-Me.     -   Embodiment 99. The trRNA of any one of embodiments 87-98,         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ terminus and three PS bonds         linking the last four nucleotides at the 3′ terminus.     -   Embodiment 100. The trRNA of embodiment 98 or 99, further         comprising 2′-O-Me or 2′-F modified nucleotides at the first         three nucleotides at the 5′ terminus, and 2′-O-Me or 2′-F         modified nucleotides at the last three nucleotides at the 3′         terminus.     -   Embodiment 101. A trRNA comprising the nucleic acids of any one         of SEQ ID Nos: 188-227.     -   Embodiment 102. A trRNA comprising nucleic acids having at least         99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, or 70%         identity to the nucleic acids of any one of SEQ ID Nos: 188-227,         wherein the modification pattern is identical to the         modification pattern shown in the reference sequence identifier.     -   Embodiment 103. The trRNA of any one of embodiments 101-102,         further comprising three phosphorothioate (PS) bonds linking the         first four nucleotides at the 5′ terminus and three PS bonds         linking the last four nucleotides at the 3′ terminus.     -   Embodiment 104. A dual guide comprising a crRNA and a trRNA,         wherein the crRNA comprises the nucleotides of any one of SEQ ID         Nos: 1-187, and wherein the trRNA comprises the nucleic acids of         any one of SEQ ID Nos: 188-227.     -   Embodiment 105. A dual guide comprising a crRNA of any one of         embodiments 55-78 and a trRNA of any one of embodiments 79-103.     -   Embodiment 106. A dual guide comprising a crRNA of any one of         embodiments 55-78 and an unmodified trRNA.     -   Embodiment 107. A dual guide comprising an unmodified crRNA and         a trRNA of any one of embodiments 79-103.

C. Modifications to terminal nucleotides

In some embodiments, the 5′ or 3′ terminal nucleotides of any of the guide RNAs described herein are modified. In some embodiments, the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in 3′ terminus region of guide RNA, including, for example, the sgRNA, the dgRNA, the crRNA, trRNA, or both crRNA and trRNA are modified. In some embodiments, the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in 3′ terminus region of guide RNA comprise more than one modification. In some embodiments, at least one of the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 3′ terminus region are modified. In some embodiments, at least two of the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in 3′ terminus region are modified. In some embodiments, at least three of the terminal (i.e., last) 1, 2, 3, 4, 5, 6, or 7 nucleotides in 3′ terminus region are modified. some embodiments, the modification comprises a PS linkage.

In some embodiments, the 5′ end of the 5′ terminus region is modified, for example, the first 1, 2, 3, 4, 5, 6, or 7 nucleotides of the sgRNA, the dgRNA, crRNA, trRNA, or both crRNA and trRNA are modified. In some embodiments, the first 1, 2, 3, 4, 5, 6, or 7 nucleotides in 3′ terminus region of guide RNA comprise more than one modification. In some embodiments, at least one of the terminal (i.e., first) 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 5′ end are modified. In some embodiments, at least two of the terminal 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 5′ end are modified. In some embodiments, at least three of the terminal 1, 2, 3, 4, 5, 6, or 7 nucleotides at the 5′ end are modified. some embodiments, the modification comprises a PS linkage.

In some embodiments, both the 5′ and 3′ termini (e.g., ends) of the guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA are modified. In some embodiments, only the 5′ terminus of the guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA is modified. In some embodiments, only the 3′ terminus of the guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA is modified.

In some embodiments, the gRNA comprises modifications at 1, 2, 3, 4, 5, 6, or 7 of the first 7 nucleotides at a 5′ end of the gRNA. In some embodiments, the gRNA comprises modifications at 1, 2, 3, 4, 5, 6, or 7 of the 7 terminal nucleotides at a 3′ end. In some embodiments, 2, 3, or 4 of the first 4 nucleotides at the 5′ end, and/or 2, 3, or 4 of the terminal 4 nucleotides at the 3′ end are modified. In some embodiments, 2, 3, or 4 of the first 4 nucleotides at the 5′ end are linked with phosphorothioate (PS) bonds.

In some embodiments, the modification to the 5′ terminus and/or 3′ terminus comprises a 2′-O-methyl (2′-O-Me) or 2′-O-(2-methoxyethyl) (2′-O-moe) modification to a nucleotide. In some embodiments, the modification comprises a 2′-fluoro (2′-F) modification to a nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) linkage between nucleotides. In some embodiments, the modification comprises an inverted abasic nucleotide. In some embodiments, the modification comprises a more than one modification selected from 2′-O-Me, 2′-O-moe, 2′-fluoro (2′-F), a phosphorothioate (PS) linkage between nucleotides, and an inverted abasic nucleotide. In some embodiments, an equivalent modification is encompassed.

In some embodiments, the guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA comprises one or more phosphorothioate (PS) linkages between the first one, two, three, four, five, six, or seven nucleotides at the 5′ terminus. In some embodiments, the guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA comprises one or more PS linkages between the last one, two, three, four, five, six, or seven nucleotides at the 3′ terminus. In some embodiments, the guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA comprises one or more PS linkages between the last one, two, three, four, five, six, or seven nucleotides at both the 5′ terminus and the 3′ terminus. In some embodiments, in addition to PS linkages, the 5′ and 3′ terminal nucleotides may comprise 2′-O-Me, 2′-O-moe, or 2′-F modified nucleotides.

In some embodiments, the guide RNA, e.g., sgRNA, dgRNA, crRNA, trRNA, or both crRNA and trRNA comprises modified nucleotides at the 5′ and 3′ terminus, and modified nucleotides in one or more other regions described in Tables 1-3 and FIG. 21A or 21C.

In some embodiments, the crRNA, trRNA, or both crRNA and trRNA comprises modified nucleotides that are not at the 5′ or 3′ ends. Specific patterns of modifications are described below and in Table 4.

3. Delivery of gRNAs and Cas Protein

In some embodiments, in addition to the at least one gRNA, the compositions provided herein further comprise a nuclease. In some embodiments, the nuclease is a Cas protein. In some embodiments, the gRNA together with a Cas protein is called a Cas RNP. In some embodiments, the Cas protein is from the Type-II CRISPR/Cas system. In some embodiments, the Cas protein is Cas9. In some embodiments, the Cas9 protein is a wild type Cas9. In some embodiments, the Cas9 protein is derived from the Streptococcus pyogenes Cas9 protein, e.g., a S. pyogenes Cas9. In some embodiments, the Cas9 protein is not derived from S. pyogenes, but functions in the same way as S. pyogenes Cas9 such that gRNA that is specific to S. pyogenes Cas9 will direct the non-S. pyogenes Cas9 to its target site. In some embodiments, the Cas induces a double strand break in target DNA. Equivalents of S. pyogenes Cas9 protein are encompassed by the embodiments described herein.

Cas9 encompasses modified and variants thereof. Modified versions of Cas9 having one catalytic domain, either RuvC or HNH, that is inactive are termed “nickases.” Nickases cut only one strand on the target DNA, thus creating a single-strand break. A single-strand break may also be known as a “nick.” In some embodiments, the compositions and methods comprise nickases. In some embodiments, the compositions and methods comprise a nickase Cas9 that induces a nick rather than a double strand break in the target DNA.

In some embodiments, the Cas protein may be modified to contain only one functional nuclease domain. For example, the Cas protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase Cas is used having a RuvC domain with reduced activity. In some embodiments, a nickase Cas is used having an inactive RuvC domain. In some embodiments, a nickase Cas is used having an HNH domain with reduced activity. In some embodiments, a nickase Cas is used having an inactive HNH domain.

In some embodiments, a conserved amino acid within a Cas protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, a Cas protein may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). In some embodiments, the Cas protein may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein).

In some embodiments, the RNP complex described herein comprises a nickase and a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNAs direct the nickase to a target sequence and introduce a double stranded break (DSB) by generating a nick on opposite strands of the target sequence (i.e., double nicking). In some embodiments, use of double nicking may improve specificity and reduce off-target effects. In some embodiments, a nickase Cas is used together with two separate guide RNAs targeting opposite strands of DNA to produce a double nick in the target DNA. In some embodiments, a nickase Cas is used together with two separate guide RNAs that are selected to be in close proximity to produce a double nick in the target DNA.

In some embodiments, chimeric Cas proteins are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease such as Fok1. In some embodiments, a Cas protein may be a modified nuclease.

In some embodiments, the Cas protein comprises a fusion protein comprising a catalytically inactive Cas9 linked to a heterologous functional domain (see, e.g., WO2014152432). In some embodiments, the catalytically inactive Cas9 is from S. pyogenes. In some embodiments, the catalytically inactive Cas9 comprises mutations that inactivate the Cas9. In some embodiments, the heterologous functional domain is a domain that modifies gene expression, histones, or DNA. In some embodiments, the heterologous functional domain is a transcriptional activation domain or a transcriptional repressor domain.

A. PAM

In some embodiments, the target sequence may be adjacent to the PAM. In some embodiments, the PAM may be adjacent to or within 1, 2, 3, or 4, nucleotides of the 3′ end of the target sequence. The length and the sequence of the PAM may depend on the Cas protein used. For example, the PAM may be selected from a consensus or a particular PAM sequence for a specific Cas9 protein or Cas9 ortholog, including those disclosed in FIG. 1 of Ran et al., Nature 520:186-191 (2015), which is incorporated herein by reference. In some embodiments, the PAM may comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NGG, NAG, NGA, NGAG, NGCG, NNGRRT, TTN, NGGNG, NG, NAAAAN, NNAAAAW, NNNNACA, GNNNCNNA, and NNNNGATT (wherein N is defined as any nucleotide, and W is defined as either A or T, and R is defined as either A or G). In some embodiments, the PAM sequence may be NGG. In some embodiments, the PAM sequence may be NGGNG. In some embodiments, the PAM sequence may be NNAAAAW.

B. Delivery of Modified gRNA

Lipid nanoparticles (LNPs) are a well-known means for delivery of nucleotide and protein cargo, and may be used for delivery of the gRNA, mRNA, Cas9, and RNPs disclosed herein. In some embodiments, the LNPs deliver nucleic acid, protein, or nucleic acid together with protein.

In some embodiments, the invention comprises a method for delivering any one of the gRNAs disclosed herein to a subject, wherein the gRNA is associated with an LNP. In some embodiments, the gRNA/LNP is also associated with a Cas9 or an mRNA encoding Cas9.

In some embodiments, the invention comprises a composition comprising any one of the gRNAs disclosed and an LNP. In some embodiments, the composition further comprises a Cas9 or an mRNA encoding Cas9.

In some embodiments, the LNPs comprise cationic lipids. In some embodiments, the LNPs comprise (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate). In some embodiments, the LNPs comprise molar ratios of a cationic lipid amine to RNA phosphate (N:P) of about 4.5.

In some embodiments, LNPs associated with the gRNAs disclosed herein are for use in preparing a medicament for treating a disease or disorder.

Electroporation is a well-known means for delivery of cargo, and any electroporation methodology may be used for delivery of any one of the gRNAs disclosed herein. In some embodiments, electroporation may be used to deliver any one of the gRNAs disclosed herein and Cas9 or an mRNA encoding Cas9.

In some embodiments, the invention comprises a method for delivering any one of the gRNAs disclosed herein to an ex vivo cell, wherein the gRNA is associated with an LNP or not associated with an LNP. In some embodiments, the gRNA/LNP or gRNA is also associated with a Cas9 or an mRNA encoding Cas9.

4. Methods of Gene Modulation

In some embodiments, the invention comprises a pharmaceutical formulation comprising any one of the gRNAs disclosed herein together with a pharmaceutically acceptable carrier. In some embodiments, the invention comprises a pharmaceutical formulation comprising any one of the gRNAs disclosed herein and an LNP together with a pharmaceutically acceptable carrier. In some embodiments, the invention comprises a pharmaceutical formulation comprising any one of the gRNAs disclosed herein, a Cas9 protein or an mRNA encoding a Cas9 protein, and a LNP together with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation is for use in preparing a medicament for treating a disease or disorder. In some embodiments, the invention comprises a method of treating a human patient comprising administering any one of the gRNAs or pharmaceutical formulations described herein.

In some embodiments, the invention comprises a method or use of modifying a target DNA comprising, administering or delivering a Cas protein or Cas mRNA and any one or more of the gRNAs disclosed herein.

In some embodiments, the invention comprises a method or use for modulation of a target gene comprising, administering or delivering a Cas protein or Cas mRNA and any one or more of the gRNAs disclosed herein. In some embodiments, the modulation is editing of the target gene. In some embodiments, the modulation is a change in expression of the protein encoded by the target gene.

In some embodiments, the method or use results in gene editing. In some embodiments, the method or use results in a double-stranded break within the target gene. In some embodiments, the method or use results in formation of indel mutations during non-homologous end joining of the DSB. In some embodiments, the method or use results in an insertion or deletion of nucleotides in a target gene. In some embodiments, the insertion or deletion of nucleotides in a target gene leads to a frameshift mutation or premature stop codon that results in a non-functional protein. In some embodiments, the insertion or deletion of nucleotides in a target gene leads to a knockdown or elimination of target gene expression. In some embodiments, the method or use comprises homology directed repair of a DSB. In some embodiments, the method or use further comprises delivering to the cell a template, wherein at least a part of the template incorporates into a target DNA at or near a double strand break site induced by the Cas protein.

In some embodiments, the method or use results in gene modulation. In some embodiments, the gene modulation is an increase or decrease in gene expression, a change in methylation state of DNA, or modification of a histone subunit. In some embodiments, the method or use results in increased or decreased expression of the protein encoded by the target gene.

In some embodiments, any of the gRNAs disclosed herein may be useful in preparing a medicament for treating a disease or disorder.

A. Measures of Gene Modulation

The efficacy of modified gRNAs can be tested in vitro and in vivo. In some embodiments, the invention comprises one or more of the gRNAs disclosed herein, wherein the gRNA results in gene modulation when provided to a cell together with Cas9. In some embodiments, the efficacy of gRNA can be measured in in vitro or in vivo assays.

1. In Vitro Measurement of Cas Efficacy

In some embodiments, the activity of a Cas RNP comprising a modified sgRNA is compared to the activity of a Cas RNP comprising an unmodified sgRNA.

In some embodiments, the activity of a Cas RNP comprising a dgRNA comprising a modified trRNA is compared to the activity of a Cas RNP comprising a dgRNA comprising an unmodified trRNA.

In some embodiments, the activity of a Cas RNP comprising a dgRNA comprising a modified crRNA is compared to the activity of a Cas RNP comprising a dgRNA comprising an unmodified crRNA.

In some embodiments, the activity of a Cas RNP comprising a dgRNA comprising a modified crRNA and a modified trRNA is compared to the activity of a Cas RNP comprising an unmodified crRNA and an unmodified trRNA.

In some embodiments, the efficiency of a gRNA in increasing or decreasing target protein expression is determined by measuring the amount of target protein. In some embodiments, the invention comprises any one of the gRNAs described herein, wherein the gRNA increases or decreases the amount of protein produced from the targeted gene. In some embodiments, the invention comprises a method of modulating protein expression comprising administering any one of the gRNAs disclosed herein to a subject, wherein the gRNA directs Cas9 to the gene encoding the target protein, and the target protein expression is increased or decreased as compared to a gRNA control that does not target Cas9 to that gene.

In some embodiments, the efficiency of editing with specific gRNAs is determined by the editing present at the target location in the genome following delivery of Cas9 and the gRNA (either sgRNA or dgRNA comprising a crRNA and trRNA). In some embodiments, the efficiency of editing with specific gRNAs is measured by next-generation sequencing. In some embodiments, the editing percentage of the target region of interest is determined. In some embodiments, the total number of sequence reads with insertions or deletions of nucleotides into the target region of interest over the total number of sequence reads is measured following delivery of a gRNA and Cas9. In some embodiments, the invention comprises a method of increasing the efficiency of gene editing comprising, administering or delivering any one of the modified gRNAs described herein to a cell, wherein the percentage of gene editing is increased as compared to a control gRNA that is not similarly modified.

In some embodiments, the efficiency of editing with specific gRNAs is measured by the presence of insertions or deletions of nucleotides introduced by successful gene editing. In some embodiments, the invention comprises a method of creating insertions or deletions of nucleotides in genes comprising, administering or delivering any one of the modified gRNAs described herein to a cell, wherein the nucleotides are inserted or deleted as compared to a control gRNA that is not similarly modified. In some embodiments, activity of a Cas9 and gRNAs is tested in biochemical assays. In some embodiments, activity of a Cas9 and gRNAs is tested in a cell-free cleavage assay. In some embodiments, activity of a Cas9 and gRNAs is tested in Neuro2A cells.

In some embodiments, Cas 9 and sgRNA or dgRNA comprising modified crRNA and/or trRNA shows similar, greater, or reduced activity compared to the unmodified sgRNA or dgRNA comprising unmodified crRNA and trRNA. In some embodiments, Cas9 and modified sgRNA or dgRNA comprising modified crRNA and/or trRNA shows enhanced activity compared to the unmodified sgRNA or dgRNA comprising unmodified crRNA and trRNA.

2. In vivo measurement of Cas efficacy

In some embodiments, the activity of modified gRNAs is measured after in vivo dosing of LNPs comprising modified gRNAs and Cas protein or mRNA encoding Cas protein.

In some embodiments, in vivo efficacy of a gRNA or composition provided herein is determined by editing efficacy measured in DNA extracted from tissue (e.g., liver tissue) after administration of gRNA and Cas9.

3. In vivo measurement of immune system activation

Modifications to gRNA as disclosed herein may reduce the subject's immune response to in vivo dosing of gRNAs. In some embodiments, activation of the subject's immune response is measured by serum concentrations of cytokine(s) following in vivo dosing of sgRNA or dgRNA comprising trRNA and crRNA together with Cas9 mRNA or protein (e.g., formulated in a LNP). In some embodiments, the cytokine is interferon-alpha (IFN-alpha), interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and/or tumor necrosis factor alpha (TNF-alpha). In some embodiments, the invention comprises a method of reducing a subject's immune response to delivery of a gRNA comprising, administering any one of the gRNAs disclosed herein, wherein the gRNA produces a reduced response by the subject's immune system following administration. In some embodiments, the invention comprises a method of reducing activation of the subject's immune system following administration as compared to a control gRNA that is not similarly modified.

In some embodiments, administration of Cas RNP or Cas9 mRNA together with the modified gRNA (e.g., sgRNA or dgRNA) produces lower serum concentration(s) of immune cytokines compared to administration of unmodified sgRNA. In some embodiments, the invention comprises a method of reducing a subject's serum concentration of immune cytokines comprising, administering any one of the gRNAs disclosed herein, wherein the gRNA produces a lower concentration of immune cytokines in a subject's serum as compared to a control gRNA that is not similarly modified.

This description and exemplary embodiments should not be taken as limiting. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about,” to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the,” and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

Examples

The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.

Example 1—Materials and Methods

A. Synthetic guide RNA (gRNA)

gRNA in both dual (dgRNA, i.e., crRNA and trRNA) and single guide (sgRNA) format were chemically synthesized by commercial vendors with modified nucleotides and linkages as provided in Table 4.

B. In vitro transcription (“IVT”) of Cas9 mRNA

Capped and polyadenylated Cas9 mRNA containing N1-methyl pseudo-U was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing a T7 promoter and a 100 nucleotide (nt) poly(A/T) region was linearized by XbaI and obtained from a commercial manufacturer. The IVT reaction to generate Cas9 modified mRNA was incubated at 37° C. for 4 hours in the following conditions: 50 ng/μL linearized plasmid; 2 mM each of GTP, ATP, CTP, and N1-methyl pseudo-UTP (Trilink); 10 mM ARCA (Trilink); 5 U/μL T7 RNA polymerase (NEB); 1 U/μL Murine RNase inhibitor (NEB); 0.004 U/μL Inorganic E. coli pyrophosphatase (NEB); and ix reaction buffer. After the 4 hr incubation, TURBO DNase (ThermoFisher) was added to a final concentration of 0.01 U/μL, and the reaction was incubated for an additional 30 minutes to remove the DNA template. The Cas9 mRNA was purified from enzyme and nucleotides using standard protocols, including silica binding columns such as a MegaClear Transcription Clean-up kit (ThermoFisher) or precipitation steps using LiCl followed by EtOH with NaOAc. The transcript concentration was determined by measuring the light absorbance at 260 nm (Nanodrop), and the transcript was analyzed by capillary electrophoresis by Bioanlayzer (Agilent).

C. Cas9 mRNA and gRNA Transfections in Neuro2A Cells

The mouse cell line Neuro2A was cultured in DMEM media supplemented with 10% fetal bovine serum and was plated at a density of 15,000 cells/well in a 96-well plate 24 hours prior to transfection. On the day of transfection, the media was aspirated from cells and replaced with fresh media. Lipofectamine-2000 (Invitrogen) was diluted 1:50 (v/v) in Opti-MEM (Invitrogen). Cas9 mRNA and single guide RNA were diluted separately in Opti-MEM. For the dual guide format, crRNA and trRNA were diluted together in 1:1 molar ratio in Opti-MEM. Both Cas9 mRNA and gRNA were mixed separately 1:1 (v/v) with diluted Lipofectamine-2000, producing two lipoplexes. After 5 minutes of incubation, lipoplexes were added in succession to cells, for a final concentration of 100 ng Cas9 mRNA/well and 0.4 μL total lipofection reagent. Guides were tested at two dose levels for each experiment, including 25 nM and 2.5 nM, 16.7 nM and 1.67 nM, 10 nM and 1 nM, 8.3 nM and 0.83 nM, and 3 nM and 0.3 nM. For dual guide, this concentration includes equimolar amounts of crRNA and trRNA, such that, for example, 25 nM crRNA and 25 nM trRNA produce 25 nM total dual guide. Cells were lysed 24 hours post transfection, and lysates were used directly in the PCR reaction that was analyzed for editing by NGS.

Cas9 mRNA with 1xNLS (SEQ ID NO: 359): GGGUCCCGCAGUCGGCGUCCAGCGGCUCUGCUUGUUCGUGUGUGUGUCGUUGCAGGCCUU AUUCGGAUCCAUGGAUAAGAAGUACUCAAUCGGGCUGGAUAUCGGAACUAAUUC CGUGGGUUGGGCAGUGAUCACGGAUGAAUACAAAGUGCCGUCCAAGAAGUUCAAGGU CCUGGGGAACACCGAUAGACACAGCAUCAAGAAAAAUCUCAUCGGAGCCCUGCUGUUUGA CUCCGGCGAAACCGCAGAAGCGACCCGGCUCAAACGUACCGCGAGGCGACGCUA CACCCGGCGGAAGAAUCGCAUCUGCUAUCUGCAAGAGAUCUUUUCGAACGAAAUGGC AAAGGUCGACGACAGCUUCUUCCACCGCCUGGAAGAAUCUUUCCUGGUGGAGGAGGA CAAGAAGCAUGAACGGCAUCCUAUCUUUGGAAACAUCGUCGACGAAGUGGCGUACCA CGAAAAGUACCCGACCAUCUACCAUCUGCGGAAGAAGUUGGUUGACUCAACUGACAA GGCCGACCUCAGAUUGAUCUACUUGGCCCUCGCCCAUAUGAUCAAAUUCCGCGGACA CUUCCUGAUCGAAGGCGAUCUGAACCCUGAUAACUCCGACGUGGAUAAGCUUUUCAU UCAACUGGUGCAGACCUACAACCAACUGUUCGAAGAAAACCCAAUCAAUGCUAGCGG CGUCGAUGCCAAGGCCAUCCUGUCCGCCCGGCUGUCGAAGUCGCGGCGCCUCGAAAA CCUGAUCGCACAGCUGCCGGGAGAGAAAAAGAACGGACUUUUCGGCAACUUGAUCGC UCUCUCACUGGGACUCACUCCCAAUUUCAAGUCCAAUUUUGACCUGGCCGAGGACGC GAAGCUGCAACUCUCAAAGGACACCUACGACGACGACUUGGACAAUUUGCUGGCACA AAUUGGCGAUCAGUACGCGGAUCUGUUCCUUGCCGCUAAGAACCUUUCGGACGCAAU CUUGCUGUCCGAUAUCCUGCGCGUGAACACCGAAAUAACCAAAGCGCCGCUUAGCGC CUCGAUGAUUAAGCGGUACGACGAGCAUCACCAGGAUCUCACGCUGCUCAAAGCGCU CGUGAGACAGCAACUGCCUGAAAAGUACAAGGAGAUCUUCUUCGACCAGUCCAAGAAUGG GUACGCAGGGUACAUCGAUGGAGGCGCUAGCCAGGAAGAGUUCUAUAAGUUCAU CAAGCCAAUCCUGGAAAAGAUGGACGGAACCGAAGAACUGCUGGUCAAGCUGAACAG GGAGGAUCUGCUCCGGAAACAGAGAACCUUUGACAACGGAUCCAUUCCCCACCAGAU CCAUCUGGGUGAGCUGCACGCCAUCUUGCGGCGCCAGGAGGACUUUUACCCAUUCCU CAAGGACAACCGGGAAAAGAUCGAGAAAAUUCUGACGUUCCGCAUCCCGUAUUACGU GGGCCCACUGGCGCGCGGCAAUUCGCGCUUCGCGUGGAUGACUAGAAAAUCAGAGGA AACCAUCACUCCUUGGAAUUUCGAGGAAGUUGUGGAUAAGGGAGCUUCGGCACAAAG CUUCAUCGAACGAAUGACCAACUUCGACAAGAAUCUCCCAAACGAGAAGGUGCUUCC UAAGCACAGCCUCCUUUACGAAUACUUCACUGUCUACAACGAACUGACUAAAGUGAAAUA CGUUACUGAAGGAAUGAGGAAGCCGGCCUUUCUGUCCGGAGAACAGAAGAAAGC AAUUGUCGAUCUGCUGUUCAAGACCAACCGCAAGGUGACCGUCAAGCAGCUUAAAGA GGACUACUUCAAGAAGAUCGAGUGUUUCGACUCAGUGGAAAUCAGCGGGGUGGAGGA CAGAUUCAACGCUUCGCUGGGAACCUAUCAUGAUCUCCUGAAGAUCAUCAAGGACAA GGACUUCCUUGACAACGAGGAGAACGAGGACAUCCUGGAAGAUAUCGUCCUGACCUU GACCCUUUUCGAGGAUCGCGAGAUGAUCGAGGAGAGGCUUAAGACCUACGCUCAUCU CUUCGACGAUAAGGUCAUGAAACAACUCAAGCGCCGCCGGUACACUGGUUGGGGCCG CCUCUCCCGCAAGCUGAUCAACGGUAUUCGCGAUAAACAGAGCGGUAAAACUAUCCU GGAUUUCCUCAAAUCGGAUGGCUUCGCUAAUCGUAACUUCAUGCAAUUGAUCCACGA CGACAGCCUGACCUUUAAGGAGGACAUCCAAAAAGCACAAGUGUCCGGACAGGGAGACUC ACUCCAUGAACACAUCGCGAAUCUGGCCGGUUCGCCGGCGAUUAAGAAGGGAAU UCUGCAAACUGUGAAGGUGGUCGACGAGCUGGUGAAGGUCAUGGGACGGCACAAACC GGAGAAUAUCGUGAUUGAAAUGGCCCGAGAAAACCAGACUACCCAGAAGGGCCAGAA AAACUCCCGCGAAAGGAUGAAGCGGAUCGAAGAAGGAAUCAAGGAGCUGGGCAGCCA GAUCCUGAAAGAGCACCCGGUGGAAAACACGCAGCUGCAGAACGAGAAGCUCUACCU GUACUAUUUGCAAAAUGGACGGGACAUGUACGUGGACCAAGAGCUGGACAUCAAUCG GUUGUCUGAUUACGACGUGGACCACAUCGUUCCACAGUCCUUUCUGAAGGAUGACUC GAUCGAUAACAAGGUGUUGACUCGCAGCGACAAGAACAGAGGGAAGUCAGAUAAUGU GCCAUCGGAGGAGGUCGUGAAGAAGAUGAAGAAUUACUGGCGGCAGCUCCUGAAUGC GAAGCUGAUUACCCAGAGAAAGUUUGACAAUCUCACUAAAGCCGAGCGCGGCGGACU CUCAGAGCUGGAUAAGGCUGGAUUCAUCAAACGGCAGCUGGUCGAGACUCGGCAGAU UACCAAGCACGUGGCGCAGAUCUUGGACUCCCGCAUGAACACUAAAUACGACGAGAA CGAUAAGCUCAUCCGGGAAGUGAAGGUGAUUACCCUGAAAAGCAAACUUGUGUCGGA CUUUCGGAAGGACUUUCAGUUUUACAAAGUGAGAGAAAUCAACAACUACCAUCACGCGCA UGACGCAUACCUCAACGCUGUGGUCGGUACCGCCCUGAUCAAAAAGUACCCUAA ACUUGAAUCGGAGUUUGUGUACGGAGACUACAAGGUCUACGACGUGAGGAAGAUGAU AGCCAAGUCCGAACAGGAAAUCGGGAAAGCAACUGCGAAAUACUUCUUUUACUCAAA CAUCAUGAACUUUUUCAAGACUGAAAUUACGCUGGCCAAUGGAGAAAUCAGGAAGAG GCCACUGAUCGAAACUAACGGAGAAACGGGCGAAAUCGUGUGGGACAAGGGCAGGGA CUUCGCAACUGUUCGCAAAGUGCUCUCUAUGCCGCAAGUCAAUAUUGUGAAGAAAAC CGAAGUGCAAACCGGCGGAUUUUCAAAGGAAUCGAUCCUCCCAAAGAGAAAUAGCGA CAAGCUCAUUGCACGCAAGAAAGACUGGGACCCGAAGAAGUACGGAGGAUUCGAUUC GCCGACUGUCGCAUACUCCGUCCUCGUGGUGGCCAAGGUGGAGAAGGGAAAGAGCAA AAAGCUCAAAUCCGUCAAAGAGCUGCUGGGGAUUACCAUCAUGGAACGAUCCUCGUU CGAGAAGAACCCGAUUGAUUUCCUCGAGGCGAAGGGUUACAAGGAGGUGAAGAAGGA UCUGAUCAUCAAACUCCCCAAGUACUCACUGUUCGAACUGGAAAAUGGUCGGAAGCG CAUGCUGGCUUCGGCCGGAGAACUCCAAAAAGGAAAUGAGCUGGCCUUGCCUAGCAA GUACGUCAACUUCCUCUAUCUUGCUUCGCACUACGAAAAACUCAAAGGGUCACCGGA AGAUAACGAACAGAAGCAGCUUUUCGUGGAGCAGCACAAGCAUUAUCUGGAUGAAAUCAU CGAACAAAUCUCCGAGUUUUCAAAGCGCGUGAUCCUCGCCGACGCCAACCUCGA CAAAGUCCUGUCGGCCUACAAUAAGCAUAGAGAUAAGCCGAUCAGAGAACAGGCCGA GAACAUUAUCCACUUGUUCACCCUGACUAACCUGGGAGCCCCAGCCGCCUUCAAGUA CUUCGAUACUACUAUCGAUCGCAAAAGAUACACGUCCACCAAGGAAGUUCUGGACGC GACCCUGAUCCACCAAAGCAUCACUGGACUCUACGAAACUAGGAUCGAUCUGUCGCA GCUGGGUGGCGAUGGCGGUGGAUCUCCGAAAAAGAAGAGAAAGGUGUAAUGAGCUAG CCAUCACAUUUAAAAGCAUCUCAGCCUACCAUGAGAAUAAGAGAAAGAAAAUGAAGA UCAAUAGCUUAUUCAUCUCUUUUUCUUUUUCGUUGGUGUAAAGCCAACACCCUGUCU AAAAAACAUAAAUUUCUUUAAUCAUUUUGCCUCUUUUCUCUGUGCUUCAAUUAAUAAAAA AUGGAAAGAACCUCGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA UCUAG Cas9 mRNA with 2xNLS and HA tag (SEQ ID NO: 360): GGGUCCCGCAGUCGGCGUCCAGCGGCUCUGCUUGUUCGUGUGUGUGUCGUUGCAGGCCUU AUUCGGAUCCAUGGAUAAGAAGUACUCAAUCGGGCUGGAUAUCGGAACUAAUUC CGUGGGUUGGGCAGUGAUCACGGAUGAAUACAAAGUGCCGUCCAAGAAGUUCAAGGU CCUGGGGAACACCGAUAGACACAGCAUCAAGAAAAAUCUCAUCGGAGCCCUGCUGUUUGA CUCCGGCGAAACCGCAGAAGCGACCCGGCUCAAACGUACCGCGAGGCGACGCUA CACCCGGCGGAAGAAUCGCAUCUGCUAUCUGCAAGAGAUCUUUUCGAACGAAAUGGCAAA GGUCGACGACAGCUUCUUCCACCGCCUGGAAGAAUCUUUCCUGGUGGAGGAGGA CAAGAAGCAUGAACGGCAUCCUAUCUUUGGAAACAUCGUCGACGAAGUGGCGUACCA CGAAAAGUACCCGACCAUCUACCAUCUGCGGAAGAAGUUGGUUGACUCAACUGACAA GGCCGACCUCAGAUUGAUCUACUUGGCCCUCGCCCAUAUGAUCAAAUUCCGCGGACA CUUCCUGAUCGAAGGCGAUCUGAACCCUGAUAACUCCGACGUGGAUAAGCUUUUCAU UCAACUGGUGCAGACCUACAACCAACUGUUCGAAGAAAACCCAAUCAAUGCUAGCGG CGUCGAUGCCAAGGCCAUCCUGUCCGCCCGGCUGUCGAAGUCGCGGCGCCUCGAAAA CCUGAUCGCACAGCUGCCGGGAGAGAAAAAGAACGGACUUUUCGGCAACUUGAUCGC UCUCUCACUGGGACUCACUCCCAAUUUCAAGUCCAAUUUUGACCUGGCCGAGGACGCGAA GCUGCAACUCUCAAAGGACACCUACGACGACGACUUGGACAAUUUGCUGGCACA AAUUGGCGAUCAGUACGCGGAUCUGUUCCUUGCCGCUAAGAACCUUUCGGACGCAAU CUUGCUGUCCGAUAUCCUGCGCGUGAACACCGAAAUAACCAAAGCGCCGCUUAGCGC CUCGAUGAUUAAGCGGUACGACGAGCAUCACCAGGAUCUCACGCUGCUCAAAGCGCU CGUGAGACAGCAACUGCCUGAAAAGUACAAGGAGAUCUUCUUCGACCAGUCCAAGAA UGGGUACGCAGGGUACAUCGAUGGAGGCGCUAGCCAGGAAGAGUUCUAUAAGUUCAUCAA GCCAAUCCUGGAAAAGAUGGACGGAACCGAAGAACUGCUGGUCAAGCUGAACAG GGAGGAUCUGCUCCGGAAACAGAGAACCUUUGACAACGGAUCCAUUCCCCACCAGAU CCAUCUGGGUGAGCUGCACGCCAUCUUGCGGCGCCAGGAGGACUUUUACCCAUUCCU CAAGGACAACCGGGAAAAGAUCGAGAAAAUUCUGACGUUCCGCAUCCCGUAUUACGU GGGCCCACUGGCGCGCGGCAAUUCGCGCUUCGCGUGGAUGACUAGAAAAUCAGAGGA AACCAUCACUCCUUGGAAUUUCGAGGAAGUUGUGGAUAAGGGAGCUUCGGCACAAAG CUUCAUCGAACGAAUGACCAACUUCGACAAGAAUCUCCCAAACGAGAAGGUGCUUCCUAA GCACAGCCUCCUUUACGAAUACUUCACUGUCUACAACGAACUGACUAAAGUGAA AUACGUUACUGAAGGAAUGAGGAAGCCGGCCUUUCUGUCCGGAGAACAGAAGAAAGC AAUUGUCGAUCUGCUGUUCAAGACCAACCGCAAGGUGACCGUCAAGCAGCUUAAAGA GGACUACUUCAAGAAGAUCGAGUGUUUCGACUCAGUGGAAAUCAGCGGGGUGGAGGA CAGAUUCAACGCUUCGCUGGGAACCUAUCAUGAUCUCCUGAAGAUCAUCAAGGACAA GGACUUCCUUGACAACGAGGAGAACGAGGACAUCCUGGAAGAUAUCGUCCUGACCUU GACCCUUUUCGAGGAUCGCGAGAUGAUCGAGGAGAGGCUUAAGACCUACGCUCAUCU CUUCGACGAUAAGGUCAUGAAACAACUCAAGCGCCGCCGGUACACUGGUUGGGGCCG CCUCUCCCGCAAGCUGAUCAACGGUAUUCGCGAUAAACAGAGCGGUAAAACUAUCCU GGAUUUCCUCAAAUCGGAUGGCUUCGCUAAUCGUAACUUCAUGCAAUUGAUCCACGACGA CAGCCUGACCUUUAAGGAGGACAUCCAAAAAGCACAAGUGUCCGGACAGGGAGA CUCACUCCAUGAACACAUCGCGAAUCUGGCCGGUUCGCCGGCGAUUAAGAAGGGAAU UCUGCAAACUGUGAAGGUGGUCGACGAGCUGGUGAAGGUCAUGGGACGGCACAAACC GGAGAAUAUCGUGAUUGAAAUGGCCCGAGAAAACCAGACUACCCAGAAGGGCCAGAA AAACUCCCGCGAAAGGAUGAAGCGGAUCGAAGAAGGAAUCAAGGAGCUGGGCAGCCA GAUCCUGAAAGAGCACCCGGUGGAAAACACGCAGCUGCAGAACGAGAAGCUCUACCU GUACUAUUUGCAAAAUGGACGGGACAUGUACGUGGACCAAGAGCUGGACAUCAAUCG GUUGUCUGAUUACGACGUGGACCACAUCGUUCCACAGUCCUUUCUGAAGGAUGACUCGAU CGAUAACAAGGUGUUGACUCGCAGCGACAAGAACAGAGGGAAGUCAGAUAAUGU GCCAUCGGAGGAGGUCGUGAAGAAGAUGAAGAAUUACUGGCGGCAGCUCCUGAAUGCGAA GCUGAUUACCCAGAGAAAGUUUGACAAUCUCACUAAAGCCGAGCGCGGCGGACU CUCAGAGCUGGAUAAGGCUGGAUUCAUCAAACGGCAGCUGGUCGAGACUCGGCAGAU UACCAAGCACGUGGCGCAGAUCUUGGACUCCCGCAUGAACACUAAAUACGACGAGAACGA UAAGCUCAUCCGGGAAGUGAAGGUGAUUACCCUGAAAAGCAAACUUGUGUCGGA CUUUCGGAAGGACUUUCAGUUUUACAAAGUGAGAGAAAUCAACAACUACCAUCACGC GCAUGACGCAUACCUCAACGCUGUGGUCGGUACCGCCCUGAUCAAAAAGUACCCUAA ACUUGAAUCGGAGUUUGUGUACGGAGACUACAAGGUCUACGACGUGAGGAAGAUGAU AGCCAAGUCCGAACAGGAAAUCGGGAAAGCAACUGCGAAAUACUUCUUUUACUCAAACAU CAUGAACUUUUUCAAGACUGAAAUUACGCUGGCCAAUGGAGAAAUCAGGAAGAG GCCACUGAUCGAAACUAACGGAGAAACGGGCGAAAUCGUGUGGGACAAGGGCAGGGA CUUCGCAACUGUUCGCAAAGUGCUCUCUAUGCCGCAAGUCAAUAUUGUGAAGAAAAC CGAAGUGCAAACCGGCGGAUUUUCAAAGGAAUCGAUCCUCCCAAAGAGAAAUAGCGACAA GCUCAUUGCACGCAAGAAAGACUGGGACCCGAAGAAGUACGGAGGAUUCGAUUC GCCGACUGUCGCAUACUCCGUCCUCGUGGUGGCCAAGGUGGAGAAGGGAAAGAGCAA AAAGCUCAAAUCCGUCAAAGAGCUGCUGGGGAUUACCAUCAUGGAACGAUCCUCGUU CGAGAAGAACCCGAUUGAUUUCCUCGAGGCGAAGGGUUACAAGGAGGUGAAGAAGGA UCUGAUCAUCAAACUCCCCAAGUACUCACUGUUCGAACUGGAAAAUGGUCGGAAGCG CAUGCUGGCUUCGGCCGGAGAACUCCAAAAAGGAAAUGAGCUGGCCUUGCCUAGCAA GUACGUCAACUUCCUCUAUCUUGCUUCGCACUACGAAAAACUCAAAGGGUCACCGGA AGAUAACGAACAGAAGCAGCUUUUCGUGGAGCAGCACAAGCAUUAUCUGGAUGAAAU CAUCGAACAAAUCUCCGAGUUUUCAAAGCGCGUGAUCCUCGCCGACGCCAACCUCGA CAAAGUCCUGUCGGCCUACAAUAAGCAUAGAGAUAAGCCGAUCAGAGAACAGGCCGA GAACAUUAUCCACUUGUUCACCCUGACUAACCUGGGAGCCCCAGCCGCCUUCAAGUACUU CGAUACUACUAUCGAUCGCAAAAGAUACACGUCCACCAAGGAAGUUCUGGACGC GACCCUGAUCCACCAAAGCAUCACUGGACUCUACGAAACUAGGAUCGAUCUGUCGCA GCUGGGUGGCGAUGGCUCGGCUUACCCAUACGACGUGCCUGACUACGCCUCGCUCGG AUCGGGCUCCCCCAAAAAGAAACGGAAGGUGGACGGAUCCCCGAAAAAGAAGAGAAA GGUGGACUCCGGAUGAGAAUUAUGCAGUCUAGCCAUCACAUUUAAAAGCAUCUCAGC CUACCAUGAGAAUAAGAGAAAGAAAAUGAAGAUCAAUAGCUUAUUCAUCUCUUUUUC UUUUUCGUUGGUGUAAAGCCAACACCCUGUCUAAAAAACAUAAAUUUCUUUAAUCAU UUUGCCUCUUUUCUCUGUGCUUCAAUUAAUAAAAAAUGGAAAGAACCUCGAGAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG

D. Primary liver hepatocytes

Primary mouse liver hepatocytes (PMH) (Gibco) were cultured per the manufacturer's protocol (Invitrogen, protocol 11.28.2012). In brief, the cells were thawed and resuspended in hepatocyte thawing medium with supplements (Gibco, Cat. CM7000) followed by centrifugation at 100 g for 10 minutes. The supernatant was discarded and the pelleted cells resuspended in hepatocyte plating medium plus supplement pack (Invitrogen, Cat. A1217601 and CM3000). Cells were counted and plated on Bio-coat collagen I coated 96-well plates (ThermoFisher, Cat. 877272) at a density of 15,000 cells/well and incubated for 5 hours at 37° C. and 5% CO2 atmosphere to allow for monolayer formation. After 5 hours, the plating media was removed and replaced with supplemented hepatocyte culture medium (Invitrogen, Cat. A1217601 and CM4000) containing LNP formulated Cas9 mRNA and guide RNA plus 3% mouse serum. LNPs were diluted from a starting dose level of 100 ng Cas9 mRNA and approximately 30 nM guide RNA per well, carrying out serial dilutions down to 0.1 ng mRNA and 0.03 nM guide per well. Cells were incubated for approximately 48 hours at 37° C. and 5% CO2 atmosphere before cell lysis and NGS analysis as described herein.

E. Lipid Nanoparticle (“LNP”) Formulation

LNPs were formulated with a cationic lipid amine to RNA phosphate (N:P) molar ratio of about 4.5. The lipid nanoparticle components were dissolved in 100% ethanol with the following molar ratios: 45 mol-% (12.7 mM) cationic lipid (e.g., (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate); 44 mol-% (12.4 mM) helper lipid (e.g., cholesterol); 9 mol-% (2.53 mM) neutral lipid (e.g., DSPC); and 2 mol-% (0.563 mM) PEG (e.g., PEG2k-DMG). The RNA cargo were prepared in 25 mM sodium acetate buffer, pH 4.5, resulting in a concentration of RNA cargo of approximately 0.45 mg/mL.

The LNPs were formed by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems NanoAssemblr™ Benchtop Instrument, according to the manufacturer's protocol. A 2:1 ratio of aqueous to organic solvent was maintained during mixing using differential flow rates.

LNP Formulation Procedure A: After mixing, the LNPs were collected, diluted in phosphate buffered saline (PBS, approximately 1:1), and then remaining buffer was exchanged into PBS (100-fold excess of sample volume), overnight at 4° C. under gentle stirring using a 10 kDa Slide-a-Lyzer™ G2 Dialysis Cassette (ThermoFisher Scientific). The LNPs were concentrated using 10 kDa Amicon spin filter (centrifugation at 4000 g at 4° C.) to achieve the desired concentration. The resulting mixture was then filtered using a 0.2 μm sterile filter. The resulting filtrate was stored at 2-8° C.

LNP Formulation Procedure B: After mixing, the LNPs were collected, diluted in 50 mM Tris at pH 7.5 (approximately 1:1), and then LNPs were exchanged into 50 mM Tris at pH 7.5 (100-fold excess of sample volume), overnight at 4° C. under gentle stirring using a 10 kDa Slide-a-Lyzer™ G2 Dialysis Cassette (ThermoFisher Scientific). The LNPs were concentrated using 10 kDa Amicon spin filter (centrifugation at 4000 g at 4° C.) to achieve twice the desired concentration. These concentrated LNPs were mixed 1:1 with 50 mM Tris, 90 mM NaCl, 10% sucrose at pH 7.5 (2×TSS). The resulting mixture was then filtered using a 0.2 μM sterile filter. The resulting filtrate was stored at −80° C.

LNP Formulation Procedure C: The RNA cargo were prepared in 25 mM sodium citrate, 100 mM sodium chloride at pH 5 resulting in a concentration of RNA cargo of approximately 0.45 mg/mL. After mixing, the LNPs were collected in water at the ratio of 3:1. The LNPs were incubated for an hour at room temperature and mixed 1:1 with water. Then they were buffer-exchanged into 1×TSS (50 mM Tris, 45 mM NaCl, 5% sucrose at pH 7.5) on PD-10 columns (GE Healthcare), using manufacturer's protocol. The LNPs were concentrated using 10 kDa Amicon spin filter (centrifugation at 4000 g at 4° C.) to achieve the desired concentration. The resulting mixture was then filtered using a 0.2 μm sterile filter. The resulting filtrate was stored at −80° C.

F. Next-Generation Sequencing (“NGS”) and Analysis for On-Target Cleavage Efficiency

To quantitatively determine the efficiency of editing at the target location in the genome, deep sequencing was utilized to identify the presence of insertions and deletions introduced by gene editing.

PCR primers were designed around the target site (e.g., TTR, FVII), and the genomic area of interest was amplified. Primer sequences are provided below in Table 5.

TABLE 5 SEQ SEQ Guide Gene Forward Primer (5′-3′) ID Reverse Primer (5′-3′) ID For experiments with guides TTR AGTCAATAATCAGAATCAGCAGGT 333 AGAAGGCACTTCTTCTTTATCTAAGGT 337 based on CR000686/G000209 targeting domains For experiments with guides TTR GTTTTGTTCCAGAGTCTATCACCG 334 ACACGAATAAGAGCAAATGGGAAC 338 based on CR000705/G000211 targeting domains For experiments with guides TTR ATTACCAGCTTAGCATCCTGTGAA 335 ACACGGTTTATAGAGCAAGAACAC 339 based on G000269/G000285 targeting domains For experiments with guides FVII AGCACATGAGACCTTCTGTTTCTC 336 GACATAGGTGTGACCCTCACAATC 340 based on CR000657/G000208 targeting domains Additional PCR was performed according to the manufacturer's protocols (Illumina) to add the necessary chemistry for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument. The reads were aligned to the human reference genome (e.g., hg38) after eliminating those having low quality scores. The resulting files containing the reads were mapped to the reference genome (BAM files), where reads that overlapped the target region of interest were selected and the number of wild type reads versus the number of reads which contain an insertion, substitution, or deletion was calculated.

The editing percentage (e.g., the “editing efficiency” or “percent editing”) is defined as the total number of sequence reads with insertions or deletions over the total number of sequence reads, including wild type.

G. LNP Delivery in vivo

CD-1 female mice, ranging 6-10 weeks of age were used in each study. Animals were weighed and grouped according to body weight for preparing dosing solutions based on group average weight. LNPs were dosed via the lateral tail vein in a volume of 0.2 mL per animal (approximately 10 mL per kilogram body weight). The animals were observed at approximately 6 hours post dose for adverse effects. Body weight was measured at twenty-four hours post-administration, and animals were euthanized at various time points by exsanguination via cardiac puncture under isoflourane anesthesia. Blood was collected into serum separator tubes or into tubes containing buffered sodium citrate for plasma as described herein. For studies involving in vivo editing, liver tissue was collected from the median lobe from each animal for DNA extraction and analysis.

H. Cytokine induction analysis

For this analysis, approximately 50-100 μL of blood was collected by tail vein nick for serum cytokine measurements. Blood was allowed to clot at room temperature for approximately 2 hours, and then centrifuged at 1000×g for 10 minutes before collecting the serum. A Luminex based magnetic bead multiplex assay (Affymetrix ProcartaPlus, catalog number Exp040-00000-801) measuring IL-6, TNF-alpha, IFN-alpha, and MCP-1 was used for cytokine analysis in collected in samples. Kit reagents and standards were prepared as directed in the manufacturer's protocol. 25 μL of mouse serum was added to wells containing 25 μL of the diluted antibody coated magnetic beads. The plate was incubated for 2 hours at room temperature and then washed. Diluted biotin antibody (50 μL) was added to the beads and incubated for 1 hour at room temperature. The beads were washed again before adding 50 μL of diluted streptavidin-PE to each well, followed by incubation for 30 minutes. The beads were washed once again and then suspended in 100 μL of wash buffer and read on the Bio-Plex 200 instrument (Bio-Rad). The data was analyzed using Bioplex Manager ver. 6.1 analysis package with cytokine concentrations calculated off a standard curve using a five parameter logistic curve fit.

I. Genomic DNA Isolation

For the in vivo studies, genomic DNA was extracted from 10 mg of tissue using a bead based extraction kit, MagMAX-96 DNA Multi-Sample Kit (ThermoFisher, Cat #4413020) according to manufacturer's protocol, which includes homogenizing the tissue in lysis buffer (approximately 400 μL/10 mg tissue). All DNA samples were normalized to 100 ng/μL concentration for PCR and subsequent NGS analysis, as described herein.

J. Transthyretin (TTR) ELISA analysis

Blood was collected and the serum was isolated as indicated. The total TTR serum levels were determined using a Mouse Prealbumin (Transthyretin) ELISA Kit (Aviva Systems Biology, Cat. OKIA00111). Kit reagents and standards were prepared according to the manufacture's protocol. Mouse serum was diluted to a final dilution of 10,000-fold with 1× assay diluent. This was done by carrying out two sequential 50-fold dilutions resulting in a 2500-fold dilution. A final 4-fold dilution step was carried out for a total sample dilution of 10,000-fold. Both standard curve dilutions (100 μL each) and diluted serum samples were added to each well of the ELISA plate pre-coated with capture antibody. The plate was incubated at room temperature for 30 minutes before washing. Enzyme-antibody conjugate (100 μL per well) was added for a 20-minute incubation. Unbound antibody conjugate was removed and the plate was washed again before the addition of the chromogenic substrate solution. The plate was incubated for 10 minutes before adding 100 μL of the stop solution, e.g., sulfuric acid (approximately 0.3 M). The plate was read on a SpectraMax M5 plate reader at an absorbance of 450 nm. Serum TTR levels were calculated by SoftMax Pro software ver. 6.4.2 using a four parameter logistic curve fit off the standard curve. Final serum values were adjusted for the assay dilution.

Example 2—Engineering Modified gRNA and In Vitro Testing

Modified gRNAs were designed in the dual guide format (dgRNA), as shown in Table 4. Accordingly, both modified crRNAs and trRNAs were designed and chemically synthesized to allow for the pairing of modified and unmodified components forming dgRNA. These pairings were transfected into Neuro2A cells at concentrations as indicated in the figures and editing efficiency (e.g., percent editing) was measured by NGS, as described in Example 1.

Certain modified crRNAs from Table 4 targeting the mouse TTR gene were transfected with Cas9 mRNA and unmodified trRNA (TR000002). Tested guides included SEQ ID Nos: 1-18. As shown in FIG. 1 , some of the modified crRNAs (together with unmodified trRNA) conferred similar or enhanced activity as compared to the unmodified control, while other modified crRNAs decreased activity.

In parallel, modified trRNAs from Table 4 were transfected with Cas9 mRNA along with an unmodified crRNA (CR000686) targeting the same sequence of the mouse TTR gene. Tested guides included SEQ ID Nos: 188-200, and 204. As shown in FIG. 2 , many of the modified trRNAs (together with unmodified crRNA) conferred similar or enhanced activity as compared to the unmodified control, while some of the modified trRNAs decreased activity.

In addition to substituting chemically modified nucleotides, some of the crRNA and trRNA pairings tested were also engineered with sequence substitutions, e.g., resulting in G-C pairings not found in the parental sequences. Tested guides included SEQ ID Nos: 15 and 201; 16 and 202; 1 and 188. As shown in FIG. 3 , one such pairing (SEQ ID Nos: 16 and 202) resulted in similar or enhanced activity as compared to the unmodified control, while two of the pairings decreased activity.

Next, pairings of modified crRNAs and modified trRNAs from Table 4 were tested. As shown in FIG. 4 , some of the pairings of modified crRNA with modified trRNA conferred similar or enhanced activity as compared to the unmodified controls, while some of the pairings decreased activity. In FIG. 4 , the column headings depict different trRNA used in the experiment, and the row headings depict different crRNA used. To determine the combination used in the experiment, you match column to row. TR000002 and CR000686 are the unmodified controls (see lower right cells).

Based on the dgRNA designs, corresponding single guide RNAs (sgRNAs) were engineered featuring aspects of some of the modified crRNAs and trRNAs, as depicted in Table 4 and FIG. 15D. These sgRNAs, SEQ ID Nos: 228-234, were also tested in Neuro2A cells, and as shown in FIG. 5 , each of the modified sgRNAs displayed activities comparable to the controls containing only 5′ and 3′ end modifications (G0000209; SEQ ID NO: 228).

A similar set of experiments were conducted for additional dgRNAs guides depicted in Table 4 and FIG. 6 . Tested guides included SEQ ID Nos: 32-47, and 1. Modified crRNAs also targeting the mouse TTR gene were transfected with Cas9 mRNA and unmodified trRNA (TR000002). As shown in FIG. 6 , some of the modified crRNAs (together with unmodified trRNA) conferred similar or enhanced activity as compared to the unmodified control (CR000686), while other modified crRNAs decreased activity.

In parallel, as shown in FIG. 7 , modified trRNAs from Table 4 were transfected with Cas9 mRNA along with an unmodified crRNA (CR000686) targeting the same sequence of the mouse TTR gene. Tested guides included SEQ ID Nos: 205-222, and 1. As shown in FIG. 7 , many of the modified trRNAs (together with unmodified crRNA) conferred similar or enhanced activity as compared to the unmodified control (TR000002), while some of the modified trRNAs decreased activity.

In addition to substituting chemically modified nucleotides, some of the crRNA and trRNA pairings tested from Table 4 were also engineered with sequence substitutions, e.g., resulting in G-C pairings or G-U mismatches (“GU wobbles”) not found in the parental sequences. As shown in FIG. 8 , some of the modifications and pairings conferred similar or enhanced activity as compared to the unmodified control, while some (e.g., the “GU wobble” or mismatch pairings) decreased activity. FIG. 8 shows results using trRNA guides shown in SEQ ID Nos: 223-227 and 188 with crRNA guides shown in SEQ ID Nos: 48-52, and 1.

Next, select pairings of the modified crRNAs and modified trRNAs from Table 4 were tested as shown in FIG. 9 . Some of the pairings of modified crRNA with modified trRNA conferred similar or enhanced activity as compared to the unmodified controls, while some of the pairings decreased activity. In FIG. 9 , the column headings depict different trRNA used in the experiment, and the row headings depict different crRNA used. To determine the combination used in the experiment, you match column to row. Unmodified controls are TR000002, and CR000686.

Some of the modified gRNAs (dgRNAs and sgRNAs) from Table 4 were also tested in a purely biochemical assay (i.e., cell free cleavage assay). Interestingly, many of the modified gRNAs that were largely inactive in the Neuro2A cells were active in the biochemical assay, indicating that such biochemical assays may not be predictive of modified gRNA activity in cells (data not shown).

Example 3. Further Testing of Modified gRNAs to Other Targets

Having established that certain modifications affected gRNA activity, it was next tested whether these modifications would affect the activity when targeting (1) a separate sequence in the same gene or (2) a sequence in a different gene. Accordingly, gRNAs targeting another sequence in the mouse TTR gene as well as a sequence in the mouse Factor-VII (FVII) gene were engineered and synthesized having certain modification patterns tested in Example 2 (see Table 4). These gRNAs were transfected into Neuro2A cells at the concentrations indicated in the figures and editing efficiency (e.g., percent editing) was measured by NGS, as described in Example 1.

Modified crRNAs from Table 4 targeting either the mouse TTR gene (different sequence as targeted in Example 2) or the mouse FVII gene, were transfected with Cas9 mRNA and unmodified trRNA (TR000002). Tested guides included those shown in FIGS. 12A and 12B. Some of the modified crRNAs (together with unmodified trRNA) conferred similar or enhanced activity as compared to the unmodified controls, while other modified crRNAs decreased activity.

In parallel, modified trRNAs from Table 4 were transfected with Cas9 mRNA along with an unmodified crRNA targeting the same sequence of the mouse TTR gene (CR000705; different sequence as targeted in Example 2) or the same sequence as the mouse FVII gene (CR000657). As shown in FIGS. 13A and 13B, many of the modified trRNAs (together with unmodified crRNAs) conferred similar or enhanced activity as compared to the unmodified controls, while some of the modified trRNAs decreased activity. This data shows that certain modification patterns tended to have similar effects over the different sequences.

Based on the dgRNA designs described above, corresponding single guide RNAs (sgRNAs) were engineered featuring aspects of some of the modified crRNAs and trRNAs. See, Table 4. These sgRNAs were also tested in Neuro2A cells. Results are shown in FIG. 10 (mouse TTR) and FIG. 11 (mouse FVII). These experiments show that some modification patterns result in similar effects even when targeting different genes.

Example 4. Testing of Modified gRNA In Vivo

Following the in vitro testing, modified sgRNAs were delivered to animals in six separate studies in order to determine whether the modifications conferred any benefits for editing in vivo.

LNPs were formulated with IVT Cas9 mRNA together with chemically modified sgRNA (targeting TTR or FVII), as described in Example 1. The ratio of mRNA:sgRNA was approximately 1:1, by weight of the RNA components. Unless otherwise indicated, the Cas9 mRNA used in the studies described in this example had the sequence of SEQ ID NO: 360 and the LNPs were formulated using LNP Formulation Procedure A described above.

In one experiment, mice (n=5 per group) were administered a single dose of LNP at 2 mg/kg and blood was collected four hours post dose for serum cytokine analysis. 7 days post dose at necropsy, livers and blood were collected for NGS measurements of editing efficiency and serum TTR analysis, respectively. Each of the sgRNAs in this experiment targeted the same sequence in the TTR gene, the only difference between the sgRNAs being the modifications made to each (See FIGS. 14A-D and 15A-E; Table 4 SEQ ID Nos: 228-234). G000209 (two lots tested) served as the less modified control, having only 2′-O-methyl modifications and phosphorothioate linkages at and between the three terminal nucleotides at both the 5′ and 3′ termini of the sgRNA, respectively. (See FIG. 15D).

The results shown in FIGS. 14A-D, show that the more heavily modified sgRNAs tended to induce less of a response for each the cytokines assayed, as compared to the less modified G000209 controls. The more heavily modified sgRNAs also conferred larger editing efficiencies in the livers of treated animals, with percent editing reaching ˜60% for two of the more heavily modified sgRNAs (e.g., G000263 and G000267) as compared to ˜44-47% for the less modified controls (G000209 lots) (FIG. 15A). Importantly, the editing efficiencies correlated with phenotypic changes as serum knockdown of TTR levels were comparable or significantly greater than the less modified controls (See e.g., G000263 and G000267 vs G000209 lots in FIGS. 15A-15B). The differences between the end-modified G000209 and highly-modified G000267 are summarized in FIGS. 15D and 15E (2′-O-Me modified nucleotides are shown in bold, and * represents phosphorothioate linkages).

In another in vivo study, three sgRNAs targeting a separate sequence in the mouse TTR gene were tested. Mice (n=5 per group) were administered a single dose of LNP at 2 mg/kg, 1 mg/kg, or 0.3 mg/kg. Blood was collected four hours post dose for serum cytokine analysis. 7 days post dose at necropsy, livers and blood were collected for NGS measurements of editing efficiency and serum TTR analysis, respectively. In this study, each of the sgRNAs targeted the same sequence in the TTR gene (a different sequence from what was targeted in the previous in vivo study) with one sgRNA being completely unmodified (G000201 (SEQ ID NO: 243)), another having only end modifications (G000211 (SEQ ID NO: 241)), with 2′-O-methyl modifications and phosphorothioate linkages at and between the three terminal nucleotides at both the 5′ and 3′ termini of the sgRNA, respectively), and a third sgRNA having the same modification pattern as G000267 in the previous in vivo study (G000282 (SEQ ID NO: 242)).

As shown in FIGS. 16A-16D, each of the sgRNAs resulted in similar responses in a dose dependent manner for each of the cytokines tested. For editing efficiency, the unmodified sgRNA (G000201 (SEQ ID NO: 243)) conferred little in vivo editing, while the heavily modified sgRNA (G000282 (SEQ ID NO: 242)) conferred levels reaching ˜60% with a dose of 2 mg/kg, which was significantly greater than the levels achieved with the less modified sgRNA (G000211 (SEQ ID NO: 241)) (FIGS. 17A and B). As with the previous in vivo study, the levels of editing correlated with the amount of serum TTR knockdown (FIGS. 17C and D).

A similar study as the second in vivo study was next conducted with another set of three sgRNAs targeting yet a different TTR sequence in the mouse TTR gene (targeting a different sequence then what was targeted in the two previous in vivo studies). Mice (n=5 per group) were administered a single dose of LNP at 2 mg/kg, 1 mg/kg, or 0.3 mg/kg. Blood was collected four hours post dose for serum cytokine analysis. 7 days post dose at necropsy, livers and blood were collected for NGS measurements of editing efficiency and serum TTR analysis, respectively. In this study, each of the sgRNAs targeted the same sequence in the TTR gene (a different sequence from what was targeted in the previous two in vivo studies) with one sgRNA being completely unmodified (G000285; (SEQ ID NO: 332)), another having only end modifications (G000269 (SEQ ID NO: 330)), with 2′-O-methyl modifications and phosphorothioate linkages at and between the three terminal nucleotides at both the 5′ and 3′ ends of the sgRNA, respectively), and a third sgRNA having the same modification pattern as G000267 and G000282 in the previous two in vivo studies (G000283 (SEQ ID NO: 331)).

In this study, the unmodified sgRNA (G000285 (SEQ ID NO: 332)) conferred little in vivo editing, while the heavily modified sgRNA (G000283 (SEQ ID NO: 331)) conferred levels reaching ˜60% with a dose of 2 mg/kg, which was significantly greater than the levels achieved with the less modified sgRNA (G000269 (SEQ ID NO: 330)) (FIGS. 18A-18B). As with the previous in vivo studies, the levels of editing correlated with the amount of serum TTR knockdown (FIG. 18C).

In a fourth in vivo study, the effects of modifications to gRNAs was evaluated for another gene (FVII). For in-study comparison, two of the sgRNAs tested in the first in vivo study were included (G000209 and G000267). Mice (n=5 per group) were administered a single dose of LNP at 2 mg/kg, 1 mg/kg, or 0.3 mg/kg, and blood was collected four hours post dose for serum cytokine analysis. 6 days post dose at necropsy, livers were collected for NGS measurements of editing efficiency. In this study, each of the sgRNAs targeted the same sequence in the TTR or FVII genes, with one sgRNA for each having only end modifications (G000208 (SEQ ID NO: 286)) for FVII, G000209 for TTR, both having 2′-O-methyl modifications and phosphorothioate linkages at and between the three terminal nucleotides at both the 5′ and 3′ ends of the sgRNA, respectively), and a second sgRNA having the same modification patterns as G000267, G000282, and G000283 in the previous in vivo studies (G000373 (SEQ ID NO: 287) for FVII; G000267 (SEQ ID NO: 234) for TTR).

As shown in FIGS. 19A-19D, each of the sgRNAs resulted in similar responses in a dose dependent manner for each of the cytokines tested. For editing efficiency, the more heavily modified sgRNA targeting FVII (G000373 (SEQ ID NO: 287)) had an increase in editing efficiency as compared to the less modified version (G000208 (SEQ ID NO: 286)) across each of the doses tested (FIG. 18A). These results were also observed for the sgRNAs targeting TTR (FIGS. 20A-20B).

In another in vivo study, ten additional sgRNAs targeting the same sequence in the mouse TTR gene as G000282 were tested. G000282 was also included in the study for comparative purposes. Mice (n=5 per group) were administered a single dose of LNP at 1 mg/kg or 0.5 mg/kg. The LNPs used in this study were formulated using LNP Formulation Procedure B described above. Seven (7) days post dose at necropsy, livers and blood were collected for NGS measurements of editing efficiency and serum TTR analysis, respectively. In this study, each of the sgRNAs targeted the same sequence in the TTR gene. The modification pattern of each sgRNA tested varied and included 2′-OMe, 2′-F, and PS modifications in the 5′ terminus, 3′ terminus, hairpin 1, hairpin 2, nexus, lower stem, bulge, and upper stem of the sgRNA. The results of this study are shown in FIGS. 22A-22C, including % editing (FIG. 22A), average editing and standard deviation (FIG. 22B), and serum TTR levels (FIG. 22C). These same sgRNAs were tested in primary mouse hepatocytes as per the methods described herein. The results of this dose response TTR editing study are shown in FIGS. 24A-24C, including % editing (FIG. 24A), dose response curves (FIG. 24B), and EC50 values (FIG. 24C).

In another in vivo study, thirteen sgRNAs targeting the same sequence in the mouse TTR gene as G000282 were tested. G000282 was also included in the study for comparative purposes. Mice (n=5 per group) were administered a single dose of LNP at 1 mg/kg. The LNPs used in this study were formulated using LNP Formulation Procedure C described above. The Cas9 mRNA used in this study had the sequence of SEQ ID NO: 359. Blood was collected four hours post dose for serum cytokine analysis. 7 days post dose at necropsy, livers and blood were collected for NGS measurements of editing efficiency and serum TTR analysis, respectively. In this study, each of the sgRNAs targeted the same sequence in the TTR gene. The sgRNAs tested include additional 2′-OMe and PS modifications in the 5′ terminus, 3′ terminus, hairpin 1, hairpin 2, and upper stem of the sgRNA. The results of this study are shown in FIGS. 23A-23C, including % editing (FIG. 23A), average % editing (FIG. 23B), and serum TTR levels (FIG. 23C). 

1-216. (canceled)
 217. A single guide RNA (sgRNA) comprising one of the following: 1) a guide RNA comprising 2′-O-Me modifications at each nucleotide in the upper stem region; or 2) a guide RNA comprising 2′-O-Me modifications at one, two, three, or four, five, six, seven, eight, nine, ten, or eleven of the following nucleotides: US1, US2, US3, US4, US5, US6, US7, US8, US9, US10, US11, and US12; or 3) a guide RNA comprising 2′-O-Me modifications at 50% or more of the nucleotides in the hairpin 1 and hairpin 2 regions; or 4) a guide RNA comprising 2′-O-Me modifications at each nucleotide in the upper stem region and 2′-O-Me modifications at 50% or more of the nucleotides in the hairpin 1 and hairpin 2 regions; or 5) a guide RNA comprising 2′-O-Me modifications at each nucleotide starting at H2-1 or H2-2 through the last nucleotide of the 3′ terminus; or 6) a guide RNA comprising a 2′-O-Me modification at LS1 and/or LS6 and no modification at LS2-LS5; or 7) a guide RNA comprising 2′-O-Me modified nucleotides at 2 or more of LS8, LS9, LS10, LS11, and/or LS12; or 8) a guide RNA comprising 2′-O-Me modifications at up to 50% of the nucleotides in the nexus region; or 9) a guide RNA comprising 2′-O-Me modifications at four or more nucleotides in the nexus region; or 10) a guide RNA comprising 2′-O-Me modified nucleotides at ten or more nucleotides in the nexus region; or 11) a guide RNA comprising 2′-O-Me modifications at 50% or more of the nucleotides in the bulge region; or 12) a guide RNA comprising 2′-O-Me modified nucleotides at three or more nucleotides in the bulge region; or 13) a guide RNA comprising the nucleotides of SEQ ID NO: 357 with the modification pattern of the guide RNA of any of 1) to 12).
 218. The sgRNA of claim 217, further comprising 2′-O-Me modifications at the first three, four, or five nucleotides at the 5′ end of the 5′ terminus.
 219. The sgRNA of claim 217, further comprising 2′-O-Me modifications at the last three, last four, or last five nucleotides at the 3′ end of the terminus region.
 220. The sgRNA of claim 219, wherein the last nucleotide at the 3′ end of the 3′ terminus region is not modified.
 221. The sgRNA of claim 219, wherein only the second to last and third to last nucleotide at the 3′ end of the 3′ terminus region are modified.
 222. The sgRNA of claim 219, wherein only the second to last, third to last, and fourth to last nucleotide in the 3′ terminus region are modified.
 223. The sgRNA of claim 217, further comprising PS bonds between the first three, four, or five nucleotides at the 5′ end of the 5′ terminus.
 224. The sgRNA of claim 223, wherein the first four nucleotides at the 5′ end of the 5′ terminus are linked with PS bonds.
 225. The sgRNA of claim 217, further comprising PS bonds between the last three, four, or five nucleotides at the 3′ end of the 3′ terminus.
 226. The sgRNA of claim 225, wherein the last four nucleotides at the 3′ end of the 3′ terminus are linked with PS bonds.
 227. The sgRNA of claim 217, wherein the first three or four nucleotides at the 5′ end of the 5′ terminus, and the last three or four nucleotides at the 3′ end of the 3′ terminus comprise 2′-O-Me modifications.
 228. The sgRNA of claim 217, wherein the first three or four nucleotides at the 5′ end of the 5′ terminus are linked with PS bonds, and the last three or four nucleotides at the 3′ end of the 3′ terminus are linked with PS bonds.
 229. The sgRNA of claim 217 wherein the first three or four nucleotides at the 5′ end of the 5′ terminus comprise 2′-O-Me modifications and are linked with PS bonds, and the last three or four nucleotides at the 3′ end of the 3′ terminus comprise 2′-O-Me modifications and are linked with PS bonds.
 230. The sgRNA of claim 217, wherein at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, of the nucleotides in the hairpin 1 and hairpin 2 regions are modified with 2′-OMe.
 231. A composition comprising the sgRNA of claim 217 and a lipid nanoparticle (LNP).
 232. A composition comprising the sgRNA of claim 217 and a nuclease or an mRNA which encodes the nuclease.
 233. A pharmaceutical formulation comprising the sgRNA of claim 217 and a pharmaceutically acceptable carrier.
 234. A method of modifying a target DNA comprising, delivering a Cas protein or a nucleic acid encoding a Cas protein, and the sgRNA of claim
 217. 235. The sgRNA of claim 217, wherein the sgRNA forms a ribonucleoprotein complex with an S. pyogenes Cas9. 